CN117157073A

CN117157073A - Methods of treating glioblastoma with aminopterin

Info

Publication number: CN117157073A
Application number: CN202280027480.3A
Authority: CN
Inventors: C·拉本德; G·C·克拉克; E·梅扎罗马; R·B·米克尔森; V·雅科夫列夫; N·史密斯
Original assignee: Ptc Medical Mp Co; Virginia Commonwealth University
Current assignee: Ptc Medical Mp Co; Virginia Commonwealth University
Priority date: 2021-02-09
Filing date: 2022-02-09
Publication date: 2023-12-01
Also published as: EP4291190A1; CA3207811A1; JP2024506336A; WO2022173834A1; US20240115572A1; IL305037A; AU2022221345A1; TW202245780A; MX2023009272A

Abstract

The application features a method of treating glioblastoma in a subject, the method comprising administering to the subject an effective amount of methotrexate or a pharmaceutically acceptable salt thereof.

Description

Methods of treating glioblastoma with aminopterin

Cross Reference to Related Applications

The present application claims the benefit and priority of pending U.S. provisional patent application serial No. 63/147,625 filed on 2/9 of 2021, the contents of which are expressly incorporated herein by reference in their entirety.

Background

Glioblastoma, also known as glioblastoma multiforme (GBM), is an invasive cancer that originates in the brain. Treatment of GBM is difficult because many drugs cannot cross the blood brain barrier and the brain is vulnerable to conventional therapies. Radiotherapy is often used in the treatment of GBM, however it often leads to side effects including fatigue, headache, memory and language problems, epilepsy, stroke-like symptoms and poor brain function. Furthermore, GBM tumors may be resistant to radiation therapy.

Aminopterin (sepiapterin) is the enzyme cofactor tetrahydrobiopterin (BH) ₄ ) Has good oral bioavailability and excellent safety profile in humans, and is currently being tested for the treatment of certain metabolic disorders. Aminopterin has also been shown to cross the blood brain barrier and to have an effect on neurotransmitter production. Once the aminopterin enters the brain, it converts to BH ₄ 。

What is needed are methods for treating GBM. What is further needed are agents useful for treating GBM that are capable of crossing the blood brain barrier. Furthermore, what is needed are methods for radiosensitizing GBM tumors. What is further needed, moreover, are radioprotectors for use in the treatment of GBM.

Disclosure of Invention

A method of treating glioblastoma in a subject is provided, comprising administering to the subject an effective amount of aminopterin or a pharmaceutically acceptable salt thereof. In some embodiments, the effective amount of the aminopterin or pharmaceutically acceptable salt thereof is from about 10mg/kg to about 60mg/kg per dose. In some embodiments, the effective amount of the aminopterin or pharmaceutically acceptable salt thereof is from about 20mg/kg to about 60mg/kg per dose. In some embodiments, the effective amount of the aminopterin or pharmaceutically acceptable salt thereof is about 20mg/kg per dose. In some embodiments, the effective amount of the aminopterin or pharmaceutically acceptable salt thereof is about 40mg/kg per dose. In some embodiments, the effective amount of the aminopterin or pharmaceutically acceptable salt thereof is about 60mg/kg per dose. In some embodiments, including any of the preceding embodiments, the effective amount of the aminopterin or pharmaceutically acceptable salt thereof is administered once daily. In some embodiments, including any of the preceding embodiments, the effective amount of the aminopterin or pharmaceutically acceptable salt thereof is administered twice daily. In some embodiments, including any of the preceding embodiments, the effective amount of the aminopterin or pharmaceutically acceptable salt thereof is administered in two equal doses. In some embodiments, including any of the preceding embodiments, an effective amount of the aminopterin or pharmaceutically acceptable salt thereof is administered with a food. In some embodiments, including any of the preceding embodiments, the administering to the subject occurs less than 30 minutes prior to or after consumption of the food. In some embodiments, including any of the preceding embodiments, the administering to the subject is performed substantially simultaneously with the food. In some embodiments, including any of the preceding embodiments, the food is a high protein and/or high fat food. In some embodiments, including any of the preceding embodiments, the food is a low fat food. In some embodiments, including any of the preceding embodiments, the food is a high calorie food. In some embodiments, including any of the preceding embodiments, the effective amount of the aminopterin or pharmaceutically acceptable salt thereof is administered in the absence of food. In some embodiments, including any of the preceding embodiments, the administering to the subject occurs more than 30 minutes prior to eating the food or more than 2 hours after eating the food. In some embodiments, including any of the preceding embodiments, the administering to the subject occurs more than 30 minutes prior to eating the food or more than 3 hours after eating the food. In some embodiments, including any of the preceding embodiments, the methotrexate or pharmaceutically acceptable salt thereof is formulated as an oral powder for suspension. In some embodiments, including any of the preceding embodiments, the methotrexate or pharmaceutically acceptable salt thereof is administered as a suspension in a flavored suspension vehicle. In some embodiments, including any of the preceding embodiments, the methotrexate or pharmaceutically acceptable salts thereof is administered as a suspension in water or fruit juice (e.g., apple, orange, grape, etc.). In some embodiments, including any of the preceding embodiments, the aminopterin or pharmaceutically acceptable salt thereof is formulated as an oral tablet, capsule, or caplet (caplet). In some embodiments, including any one of the preceding embodiments, the method further comprises treating the subject with therapeutic radiation. In some embodiments, including any of the preceding embodiments, the method further comprises administering the additional therapeutic agent to the subject in need thereof. In some embodiments, including any of the preceding embodiments, the method further comprises administering the additional therapeutic agent to the subject in need thereof. In some embodiments, including any of the preceding embodiments, the method further comprises administering the additional therapeutic agent to the subject in need thereof. In some embodiments, including any of the preceding embodiments, the method comprises administering the methotrexate or pharmaceutically acceptable salt thereof for at least 5 days. In some embodiments, including any of the preceding embodiments, the method comprises administering the methotrexate or pharmaceutically acceptable salt thereof for at least 6 days. In some embodiments, including any of the preceding embodiments, the method comprises administering the methotrexate or pharmaceutically acceptable salt thereof for at least 10 days. In some embodiments, including any of the preceding embodiments, the method comprises administering the methotrexate or pharmaceutically acceptable salt thereof for at least 14 days. In some embodiments, including any of the preceding embodiments, the method comprises administering the methotrexate or pharmaceutically acceptable salt thereof for at least 15 days. In some embodiments, including any of the preceding embodiments, the method comprises administering the methotrexate or pharmaceutically acceptable salt thereof for at least 30 days. In some embodiments, including any of the preceding embodiments, the method further comprises administering the methotrexate or pharmaceutically acceptable salt thereof for at least 6 days, or at least 10 days, or at least 14 days, or at least 30 days prior to the therapeutic radiation. In some embodiments, including any of the preceding embodiments, the method further comprises administering the aminopterin or pharmaceutically acceptable salt thereof for at least 6 days, or at least 10 days, or at least 14 days, or at least 30 days after the therapeutic radiation. In some embodiments, including any of the preceding embodiments, the method further comprises administering the additional therapeutic radiation to the subject in a continuous and simultaneous manner. In some embodiments, including any of the preceding embodiments, the method comprises administering the aminopterin or pharmaceutically acceptable salt thereof daily for a treatment period of 28 days. In some embodiments, including any of the preceding embodiments, the method further comprises administering the methotrexate or pharmaceutically acceptable salt thereof daily for a continuous 28-day treatment period. In some embodiments, including any of the preceding embodiments, the method further comprises administering the aminopterin or pharmaceutically acceptable salt thereof daily for at least six 28-day treatment cycles. In some embodiments, including any of the preceding embodiments, the administration of the aminopterin or pharmaceutically acceptable salt thereof is continued daily for a continuous 28-day treatment period until disease Progression (PD) occurs. In some embodiments, including any of the preceding embodiments, the administration of the aminopterin or pharmaceutically acceptable salt thereof is continued daily for a continuous 28-day treatment period until death. In some embodiments, including any one of the preceding embodiments, the method further comprises treating the subject with Temozolomide (TMZ). In some embodiments, including any one of the preceding embodiments, the subject is a human.

In some embodiments of any of the methods described herein, administration of the aminopterin or pharmaceutically acceptable salt thereof results in at least 50ng/mL (e.g., at least 60ng/mL, at least 100ng/mL, at least 200ng/mL, at least 400ng/mL, at least 600ng/mL, at least 1000ng/mL, or at least 2000ng/mL, or 50ng/mL to 100ng/mL, 60ng/mL to 400ng/mL, 200ng/mL to 600ng/mL, 400ng/mL to 1000ng/mL, or 600ng/mL to)1500 ng/mL) BH ₄ Concentration.

In some embodiments of any of the methods described herein, the effective amount is sufficient to produce a BH of at least 50ng/mL (e.g., at least 60ng/mL, at least 100ng/mL, at least 200ng/mL, at least 400ng/mL, at least 600ng/mL, at least 1000ng/mL, or at least 2000ng/mL, or 50ng/mL to 100ng/mL, 60ng/mL to 400ng/mL, 200ng/mL to 600ng/mL, 400ng/mL to 1000ng/mL, 600ng/mL to 1500 ng/mL) in the plasma of the subject within 10 hours of administration of the aminopterin or a pharmaceutically acceptable salt thereof ₄ Concentration amounts (e.g., 2.5mg/kg to 100mg/kg per dose).

In some embodiments of any of the methods described herein, the effective amount of the aminopterin or pharmaceutically acceptable salt thereof is about 2.5mg/kg to 100mg/kg (e.g., about 20mg/kg to about 60mg/kg, or about 20mg/kg, about 30mg/kg, about 40mg/kg, about 50mg/kg, about 60 mg/kg) per dose.

In some embodiments of any of the methods described herein, an effective amount of the methotrexate or pharmaceutically acceptable salt thereof is administered with food. In some embodiments of any of the methods described herein, the effective amount is sufficient to produce a BH of at least 50ng/mL (e.g., at least 60ng/mL, at least 100ng/mL, at least 200ng/mL, at least 400ng/mL, at least 600ng/mL, at least 1000ng/mL, or at least 2000ng/mL, or 50ng/mL to 100ng/mL, 60ng/mL to 400ng/mL, 200ng/mL to 600ng/mL, 400ng/mL to 1000ng/mL, or 600ng/mL to 1500 ng/mL) in the subject's plasma within 10 hours of administration with the food ₄ Concentration amounts (e.g., 1mg/kg to 100mg/kg per dose, or 2.5mg/kg to 100mg/kg per dose). In some embodiments, an effective amount comprises a maximum BH in the plasma of the subject sufficient to produce at least 50ng/mL (e.g., at least 60ng/mL, at least 100ng/mL, at least 200ng/mL, at least 400ng/mL, at least 600ng/mL, at least 1000ng/mL, or at least 2000ng/mL, or 50ng/mL to 100ng/mL, 60ng/mL to 400ng/mL, 200ng/mL to 600ng/mL, 400ng/mL to 1000ng/mL, or 600ng/mL to 1500 ng/mL) than within 10 hours of administration of the aminopterin or pharmaceutically acceptable salt thereof without food ₄ The dose of plasma concentration (Cmax) is at least 5% (at least 10%, at least 20%, at least 50%, to70%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150%) less dose.

In some embodiments of any of the methods described herein, the administering to the subject occurs less than 30 minutes prior to or after eating the food, e.g., up to 1 hour immediately prior to or after eating the food. In some embodiments, the administration to the subject is substantially concurrent with the food. In some embodiments of any of the methods described herein, the food is a high protein food. In some embodiments of any of the methods described herein, the food is a high fat food (e.g., at least 25%, 30%, 40%, or 50% of the calories are from fat). In some embodiments of any of the methods described herein, the food is a high protein and high fat food. In some embodiments, the food is a high calorie food (e.g., the food comprises at least 100 calories, such as at least 200 calories, at least 300 calories, at least 400 calories, at least 500 calories, such as 500-1500 or 800-1000 calories). In some embodiments of any of the methods described herein, the food is a meal, such as breakfast, lunch or dinner. In some embodiments of any of the methods described herein, the food is a low fat food (e.g., no more than 25% of calories from fat).

In some embodiments of any of the methods described herein, administration with the food (e.g., less than 30 minutes before eating the food or after eating the food, e.g., up to 1 hour before eating the food) results in BH compared to administration without the food (e.g., more than 2 hours after eating the food until 30 minutes before eating other foods) ₄ Is increased (e.g., by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150%).

In some embodiments of any of the methods described herein, and in the absence of foodAdministration of the substance (e.g., less than 30 minutes prior to consumption of the food or after consumption of the food, e.g., up to 1 hour after consumption of the food) results in BH when administered with the food (e.g., less than 30 minutes prior to consumption of the food or after consumption of the food, e.g., up to 1 hour after consumption of the food) ₄ And the extent of production of (c) and the resulting plasma exposure (AUC _0-last ) An increase (e.g., at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150%).

In some embodiments of any of the methods described herein, the methotrexate or pharmaceutically acceptable salt thereof is provided in a composition separate from the food being consumed (e.g., the methotrexate or pharmaceutically acceptable salt thereof is not incorporated into the food product). In some embodiments of any of the methods described herein, the edible food occurs prior to administration of the aminopterin or pharmaceutically acceptable salt thereof (e.g., the edible food occurs between 1 hour prior to administration of the aminopterin or pharmaceutically acceptable salt thereof and immediately prior to administration of the aminopterin or pharmaceutically acceptable salt thereof). In some embodiments of any of the methods described herein, the food product occurs after administration of the aminopterin or pharmaceutically acceptable salt thereof (e.g., the food product occurs between about immediately after administration and 30 minutes after administration).

In some embodiments of any of the foregoing methods, the effective amount is an amount (e.g., 2.5mg/kg to 100mg/kg per dose) sufficient to produce a maximum plasma concentration (Cmax) of at least 0.5ng/mL (e.g., at least 1ng/mL, at least 1.5ng/mL, at least 2.5ng/mL, or at least 3.5 ng/mL) in the subject's plasma within 1 hour of administration without food, e.g., the effective amount comprises a dose at least 10% (e.g., at least 20%, at least 40%, at least 60%, to 60%) lower in the subject's plasma than the maximum plasma concentration (Cmax) sufficient to produce at least 0.5ng/mL (e.g., at least 1ng/mL, at least 1.5ng/mL, at least 2.5ng/mL, or at least 3.5 ng/mL) in the subject's plasma within 1 hour of administration of the aminopterin with food 80%, at least 100%, or at least 120%) less dose. In some embodiments, administration (e.g., occurs more than about 30 minutes before eating the food or at least 2 hours after eating the food) results in an increase (e.g., at least 10% (at least 20%, at least 40%, at least 60%, at least 80%, at least 100%, or at least 120%) in the maximum plasma, CSF, and/or brain concentration (Cmax) of the aminopterin as compared to administration with the food (e.g., occurs less than 30 minutes before eating the food, 2 hours after eating the food). In some embodiments, administration (e.g., occurs more than 30 minutes before eating the food or at least 2 hours after eating the food) results in a degree of absorption of the Aminopterin (AUC) compared to administration with the food (e.g., administration to a subject occurs less than 30 minutes before eating the food, less than 2 hours after eating the food) _0-last ) An increase (e.g., of at least 10% (at least 20%, at least 40%, at least 60%, at least 80%, at least 100%, or at least 120%)).

In some embodiments of any of the methods described herein, the effective amount of the aminopterin or pharmaceutically acceptable salt thereof is administered in two equal doses (e.g., two doses at different times of the day). In some embodiments of any of the methods described herein, an effective amount of the methotrexate or pharmaceutically acceptable salt thereof is administered once daily. In some embodiments of any of the methods described herein, the effective amount of the aminopterin or pharmaceutically acceptable salt thereof is administered in two 60mg/kg doses (e.g., one 60mg/kg dose in the morning and one 60mg/kg dose in the evening). In some embodiments of any of the methods described herein, the effective amount of the aminopterin or pharmaceutically acceptable salt thereof is administered in two 40mg/kg doses (e.g., one 40mg/kg dose in the morning and one 40mg/kg dose in the evening). In some embodiments of any of the methods described herein, the effective amount of the aminopterin or pharmaceutically acceptable salt thereof is administered in two 30mg/kg doses (e.g., one 30mg/kg dose in the morning and one 30mg/kg dose in the evening). In some embodiments of any of the methods described herein, the effective amount of the aminopterin or pharmaceutically acceptable salt thereof is administered in two 20mg/kg doses (e.g., one 20mg/kg dose in the morning and one 20mg/kg dose in the evening). In some embodiments of any of the methods described herein, the effective amount of the aminopterin or pharmaceutically acceptable salt thereof is administered in two 10mg/kg doses (e.g., one 10mg/kg dose in the morning and one 10mg/kg dose in the evening).

In an embodiment of any one of the methods described herein, the method comprises administering to the subject an effective amount of methotrexate or a pharmaceutically acceptable salt thereof once daily with food. In embodiments of any of the methods described herein, the method comprises administering to the subject an effective amount of the methotrexate, or pharmaceutically acceptable salt thereof, more than once daily (e.g., twice daily) with food. In an embodiment of any one of the methods described herein, the method comprises administering to the subject an effective amount of methotrexate, or a pharmaceutically acceptable salt thereof, once daily in the absence of food. In embodiments of any of the methods described herein, the method comprises administering to the subject an effective amount of methotrexate, or a pharmaceutically acceptable salt thereof, more than once per day (e.g., twice per day) in the absence of food.

In some embodiments of any of the methods described herein, the subject is a child (e.g., the subject is less than 18 years old, less than 17 years old, less than 16 years old, less than 15 years old, less than 14 years old, less than 13 years old, less than 12 years old, less than 11 years old, less than 10 years old, less than 9 years old, less than 8 years old, less than 7 years old, less than 6 years old, less than 5 years old, less than 4 years old, less than 3 years old, less than 2 years old, less than 1 year old). In some embodiments of any of the methods described herein, the subject is an adult (e.g., the subject is older than 18 years). In some embodiments, the subject is older than 20 years, older than 30 years, older than 40 years, older than 50 years, older than 60 years, older than 7 years, older than 80 years, older than 90 years.

In some embodiments of any of the methods described herein, the methotrexate or pharmaceutically acceptable salt thereof is formulated as an oral powder for suspension. In some embodiments of any of the methods described herein, the method comprises administering to the subject in need thereof, a suspension of methotrexate or a pharmaceutically acceptable salt thereof (e.g.Oral Mix). In some embodiments of any of the methods described herein, the methotrexate or pharmaceutically acceptable salt thereof is administered as a suspension in water or fruit juice (e.g., apple juice). In some embodiments of any of the methods described herein, the methotrexate or pharmaceutically acceptable salt thereof is administered as a suspension in a food (such as applesauce or pudding). In some embodiments of any of the methods described herein, the methotrexate or pharmaceutically acceptable salt thereof is formulated as a tablet, capsule, or caplet.

Definition of the definition

In the present application, unless the context clearly indicates otherwise, (i) the term "a" or "an" may be understood to mean "at least one"; (ii) the term "or" may be understood to mean "and/or"; (iii) The terms "comprising" and "including" are to be construed as encompassing the listed components or steps individually as well as in combination with one or more additional components or steps; and (iv) the term "about" may be understood as allowing standard deviation, as will be appreciated by one of ordinary skill in the art; and (v) where ranges are provided, endpoints are included.

It will be understood that the descriptions of compounds, compositions, formulations, and methods of treatment described herein include "comprising," "consisting of … …," and "consisting essentially of … …" embodiments. In some embodiments, for all compositions described herein and all methods of using the compositions described herein, the compositions may comprise, or "consist essentially of, the listed components or steps. When a composition is described as "consisting essentially of" the listed components, the composition contains the listed components and may contain other components that do not substantially affect the condition being treated, but does not contain any other components that substantially affect the condition being treated other than those specifically listed; alternatively, if the composition does contain additional components other than those listed that substantially affect the condition being treated, the composition does not contain sufficient concentrations or amounts of additional components to substantially affect the condition being treated. When a method is described as "consisting essentially of the recited steps, the method contains the recited steps and may contain other steps that do not substantially affect the condition being treated, but the method does not contain any other steps that substantially affect the condition being treated other than those specifically recited. As a non-limiting specific example, when a composition is described as "consisting essentially of one component," the composition may additionally contain any amount of a pharmaceutically acceptable carrier, vehicle, or diluent as well as other such components that do not substantially affect the condition being treated.

All references to aminopterin contained herein refer to either aminopterin or a pharmaceutically acceptable salt of the same unless the context clearly indicates otherwise.

As used herein, the term "about" means a value within ±10% of the value following the term "about". References herein to "about" a value or parameter include (and describe) variations with respect to the value or parameter itself. For example, a description referring to "about X" includes a description of "X".

As used herein, the term "administering" refers to administering a composition (e.g., a compound or formulation comprising a compound as described herein) to a subject or system. Administration to an animal subject (e.g., to a human) can be by any suitable route. For example, in some embodiments, administration may be bronchial (including by bronchial instillation), buccal, enteral, intradermal, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including by intratracheal instillation), transdermal, vaginal, and vitreous.

The "effective amount" of a compound may vary depending on factors such as: disease state, age, sex and weight of the individual, and ability of the compound to elicit a desired response. A therapeutically effective amount encompasses an amount in which the therapeutically beneficial effect of a compound exceeds any toxic or detrimental effect thereof. An effective amount also encompasses an amount sufficient to confer a benefit (e.g., a clinical benefit).

As used herein, the term "food" refers to a solid food having sufficient volume and fat content not to be rapidly dissolved and absorbed in the stomach. For example, a meal, such as breakfast, lunch or dinner. As used herein, the term "with food" refers to administering the composition between about 30 minutes before a meal to about two hours after a meal, for example. The terms "without food," "fasted," or "fasting" refer to a condition in which no solid food is consumed for about 30 minutes after at least about 2 hours (e.g., at least 3 hours) of consumption of other solid food until before the consumption of other solid food.

"low fat food" indicates a meal from fat that does not exceed 25% of calories. In some embodiments, the low fat meal contains about 11-14g fat. In some embodiments, the low fat food contains about 400-500 total calories.

"native protein" means a protein from a natural source (e.g., animal, plant, or fungal).

As used herein, the term "pharmaceutical composition" refers to a composition containing a compound described herein formulated with pharmaceutically acceptable excipients. The pharmaceutical composition may be formulated, for example, for oral administration in unit dosage form (e.g., tablet, capsule, caplet, gelatin soft capsule (gel cap), suspension, solution, or syrup); for topical administration (e.g., as a cream, gel, lotion, or ointment); for intravenous administration (e.g., as a sterile solution without particulate emboli (particulate emboli) and in a solvent system suitable for intravenous use); or in any other pharmaceutically acceptable formulation.

As used herein, the term "pharmaceutically acceptable salt" means any salt that is suitable for use in contact with human and animal tissue without undue toxicity, irritation, allergic response, and commensurate with a reasonable benefit/risk ratio, within the scope of sound medical judgment. Pharmaceutically acceptable salts are well known in the art. Pharmaceutically acceptable salts are described, for example, in Berge et al, J.pharmaceutical Sciences 66:1-19,1977 and Pharmaceutical Salts: properties, selection, and Use, (P.H.Stahl and C.G.Wermuth et al), wiley-VCH, 2008. Salts may be prepared in situ during the final isolation and purification of the compounds described herein, or separately by reacting the free base groups with a suitable organic acid.

Frequently, the compounds are prepared or used as pharmaceutically acceptable salts prepared as addition products of pharmaceutically acceptable acids. Suitable pharmaceutically acceptable acids and methods for preparing the appropriate salts are well known in the art. Salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids.

Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate (benzenesulfonate), benzoate, benzenesulfonate (besylate), bisulfate, borate, butyrate, camphorite, camphorsulfonate, citrate, cyclopentapropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, gentisate, glucoheptonate, glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, pamoate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, and valerate.

As used herein, the term "substantially free" refers to qualitative conditions that exhibit complete or near complete absence of a compound or range or degree of compound type of interest. Those of ordinary skill in the biological arts will appreciate that little, if any, biological and chemical phenomena can be unambiguously determined to be zero, for example, due to inherent errors in any measurement. Thus, the term "substantially free" is used herein to capture the potential lack of integrity inherent in many biological and chemical measurements.

As used herein, the term "subject" or "participant" or "patient" refers to any organism to which a compound or composition according to the invention may be administered, e.g., for experimental, diagnostic, prophylactic and/or therapeutic purposes. Typical subjects include any animal (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans). The subject may seek or need treatment, require treatment, be receiving treatment in the future, or be a person or animal that is being cared for by a trained professional for a particular disease or condition.

As used herein, the terms "treatment", "treatment" or "treatment" mean both therapeutic and prophylactic or preventative (prophylactic) measures, wherein the object is to prevent or slow down (lessen) an undesired physiological condition, disorder or disease, or to obtain a beneficial or desired clinical result. Beneficial or desired clinical results include, but are not limited to: alleviation of symptoms; a reduction in the extent of a condition, disorder or disease; a stable (i.e., non-worsening) state of a condition, disorder or disease; delayed onset or slowing of the progression of a condition, disorder or disease; improvement or alleviation (whether partial or complete), whether detectable or undetectable, of a condition, disorder or disease state; the improvement in the at least one measurable physical parameter is not necessarily perceived by the subject; enhancement or improvement of a condition, disorder or disease. Treatment involves eliciting a clinically significant response without undue levels of side effects. Treatment also includes prolonging survival compared to expected survival without treatment.

"disease Progression (PD)" indicates one or more of the following: (1) The occurrence of one or more new lesions, (2) at least a 20% increase in the sum of target lesion diameters referenced to the minimum sum in the study (including the baseline sum if the baseline sum is the minimum in the study); in addition to a relative increase of 20%, the sum must also exhibit an absolute increase of at least 5 mm. Eisenhauer EA, therasse P, bogaerts J et al New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur JCancer.2009;45:228-247.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials for use in the present disclosure are described herein; in addition, suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

The details of one or more embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and from the claims.

Drawings

Fig. 1 is a graph showing survival of nude (U87) mice with glioblastoma treated with either aminopterin in combination with irradiation or irradiation alone, as compared to a control.

Fig. 2 is a graph showing survival of syngeneic (GL 261) mice treated with Sepiolite (SP), sepiolite in combination with radiation (IR), or with radiation, as compared to a control.

Fig. 3 is a graph showing survival of mice with glioblastoma treated with Temozolomide (TMZ) alone or with aminopterin, as compared to a control.

FIG. 4 is a graph showing the relative to the control (naive) Graph of New Object Recognition (NOR) discrimination index for mice treated with radiation or radiation and aminopterin.

Fig. 5 is a schematic of a phase 2 study of muropterin in combination with temozolomide and therapeutic radiation in a newly diagnosed or recurrent glioblastoma.

Detailed Description

The inventors have found that methotrexate is effective in treating patients with glioblastoma. In some embodiments, the aminopterin is administered with food. In some embodiments, the methotrexate is administered in the absence of food. Without wishing to be bound by theory, the epstein may not only sensitize glioblastoma to therapeutic radiation and/or chemotherapeutic agents, but may also protect the brain from side effects from therapeutic radiation and/or chemotherapeutic agents. In some embodiments, the methotrexate prevents, delays and/or reduces cognitive decline due to therapeutic radiation and/or chemotherapeutic agents. Accordingly, the invention features methods for treating glioblastoma in a subject by administering methotrexate or a pharmaceutically acceptable salt thereof. The methods herein may further comprise treatment with a chemotherapeutic agent and/or therapeutic radiation.

Murrain pterin

Aminopterin ((S) -2-amino-6- (2-hydroxypropionyl) -7, 8-dihydropterin-4 (3H) -one) enters the cell and is converted to 7, 8-dihydrobiopterin by the enzyme aminopterin reductase. 7, 8-dihydrobiopterin is subsequently converted to BH via reduction by dihydrofolate reductase ₄ 。

The aminopterin or a pharmaceutically acceptable salt thereof may be formulated in a pharmaceutical composition. In some embodiments, the pharmaceutical compositions of the invention comprise 20-30% of the aminopterin or salt thereof, e.g., about 20%, 22%, 25%, 27% or 30% by total weight. In some embodiments, the pharmaceutical composition comprises greater than 20% of the total weight of the aminopterin, e.g., greater than 25%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%. In some embodiments, the pharmaceutical composition comprises less than 20% of the aminopterin, e.g., less than 20%, less than 15%, less than 10%, or less than 5% by total weight.

In some embodiments, the invention features a pharmaceutical composition that includes methotrexate or a pharmaceutically acceptable salt thereof and less than 10% antioxidant, e.g., about 9%, 7%, 5%, 3%, 1%, 0.5%, 0.25%, 0.1%, or no antioxidant, by total weight. The antioxidant may be ascorbic acid. In some embodiments, the ratio of the aminopterin or pharmaceutically acceptable salt thereof to the antioxidant is 1:1, or greater than 1:1, such as 2:1, 5:1, 7:1, or 10:1 by weight. The pharmaceutical composition may comprise 20-30% of the total weight of the aminopterin or a pharmaceutically acceptable salt thereof, e.g., about 20%, 22%, 25%, 27% or 30%. The pharmaceutical composition may further comprise a dispersing agent, such as croscarmellose sodium. The pharmaceutical composition may comprise 0.1-1.5% of the dispersing agent, e.g. 0.1%, 0.5%, 1% or 1.5% by total weight. In some embodiments, the pharmaceutical composition comprises at least one anti-caking agent, such as colloidal silicon dioxide or microcrystalline cellulose. The pharmaceutical composition may include 65-75% of the anti-caking agent, for example about 65%, 67%, 70%, 73% or 75% by total weight. In some embodiments, the pharmaceutical composition includes both colloidal silica and microcrystalline cellulose. In some embodiments, the pharmaceutical composition comprises 60-65% microcrystalline cellulose by total weight and 5-7% colloidal silicon dioxide by total weight. In some embodiments, the crystalline form of the aminopterin is formulated as particles having a size of less than 140 μm, for example, about 120 μm, 110 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 20 μm, 10 μm, or 5 μm. In some embodiments, the pharmaceutical composition includes less than 1% impurities, such as lactoylpterin (lactoylpterin), by total weight, e.g., the composition includes less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, or less than 0.2%.

In some embodiments, the aminopterin is a salt of aminopterin with, for example, sulfuric acid, p-toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid, malonic acid, tartaric acid (e.g., L-tartaric acid), phosphoric acid, gentisic acid, fumaric acid, glycolic acid, acetic acid, or nicotinic acid.

In some embodiments, the methotrexate or pharmaceutically acceptable salt thereof is in a crystalline form. The crystalline form of the crystalline aminopterin free base or salt of the aminopterin may exist as an anhydrate (e.g., without any bound water or solvent or hydration or solvation), or as a hydrate, partial hydrate (e.g., hemihydrate, sesquihydrate, etc.), as a dihydrate, trihydrate, or the like, wherein the crystalline form binds hydrated water or solvent molecules associated with the crystalline form of the aminopterin or salt thereof. In one embodiment, the crystalline aminopterin is present as a monohydrate or as a hemihydrate.

Exemplary salts, co-crystals, and crystalline forms of aminopterin are described in WO 2018/102314, WO 2018/102315, WO 2019/232120, and WO 2019/046849, the crystalline forms, salts, and co-crystals of which are incorporated herein by reference in their entirety.

In some embodiments, the crystalline form of the epstein free base is crystalline form F of the epstein free base and is characterized by an X-ray powder diffraction pattern obtained by irradiation with Cu ka X-rays having peaks expressed in 2θ at 9.7 ° ± 0.5 (e.g., 9.7 ° ± 0.2), 10.2 ° ± 0.5 (e.g., 10.2 ° ± 0.2) and 11.3 ° ± 0.5 (e.g., 11.3 ° ± 0.2). In other embodiments, the crystalline form of aminopterin is characterized by an X-ray powder diffraction pattern obtained by irradiation with Cu ka X-rays having peaks expressed in 2θ at: 9.7°±0.5 (e.g., 9.7°±0.2), 10.2°±0.5 (e.g., 10.2°±0.2), 11.3°±0.5 (e.g., 11.3°±0.2), 14.0°±0.5 (e.g., 14.0°±0.2), 14.6°±0.5 (e.g., 14.6°±0.2), 19.9°±0.5 (e.g., 19.9°±0.2), 22.2°±0.5 (e.g., 22.2°±0.2), 25.3°±0.5 (e.g., 25.3°±0.2), and 32.4°±0.5 (e.g., 32.4°±0.2). In a substantially pure form of this crystalline form, peaks can be observed at the angle of refraction 2θ as described in table 1. Alternatively or in addition, the crystalline form is characterized by a DSC curve showing two endotherms at 71.6 ℃ and 233.4 ℃.

TABLE 1

Position [2 theta ]](+ -0.5, e.g. + -0.2)	Relative intensity
		9.7	98.27
10.2	100.00
		11.3	22.47
14.0	5.01
		14.6	12.36
19.9	5.63
		21.1	3.72
22.2	5.37
		22.7	4.04
24.5	2.99
		25.3	17.65
27.2	3.10
		32.4	5.29
36.7	2.72

In some embodiments, the crystalline form of the aminopterin free base is crystalline form B of the aminopterin free base and has peaks at diffraction angles 2θ (°) of 8.4 ° ± 0.5 (e.g., 8.4 ° ± 0.2), 16.9 ° ± 0.5 (e.g., 16.9 ° ± 0.2), and 25.4 ° ± 0.5 (e.g., 25.4 ° ± 0.2), as measured by or calculated from X-ray diffraction with Cu ka X-ray irradiation. In some embodiments, crystalline form B of the aminopterin free base has peaks at diffraction angles 2θ (°) of 8.4 ° ± 0.5 (e.g., 8.4 ° ± 0.2), 14.9 ° ± 0.5 (e.g., 14.9 ° ± 0.2), 16.9 ° ± 0.5 (e.g., 16.9 ° ± 0.2), 25.4 ° ± 0.5 (e.g., 25.4 ° ± 0.2), and 34.1 ° ± 0.5 (e.g., 34.1 ° ± 0.2) as measured by or calculated from X-ray diffraction with Cu ka X-ray irradiation. In this crystalline form of the substantially pure material, peaks can be observed at the angle of refraction 2θ as described in table 2. Alternatively or in addition, the crystalline form is characterized by a DSC profile showing a melting event at 195.2 ℃.

TABLE 2

Position [2 theta ]](+ -0.5, e.g. + -0.2)	Relative intensity
		8.4	100.00
14.9	2.34
		16.9	10.70
25.4	84.90
		34.1	3.00

In some embodiments, the crystalline form of the aminopterin free base is crystalline form C of the aminopterin free base and has peaks at diffraction angles 2θ (°) of 5.7 ° ± 0.5 (e.g., 5.7 ° ± 0.2), 7.8 ° ± 0.5 (e.g., 7.8 ° ± 0.2), and 25.4 ° ± 0.5 (e.g., 25.4 ° ± 0.2), as measured by or calculated from X-ray diffraction with Cu ka X-ray irradiation. In some embodiments, crystalline form C of the seppterin free base has peaks at diffraction angles θ (°) of 5.7 ° ± 0.5 (e.g., 5.7 ° ± 0.2), 7.8 ° ± 0.5 (e.g., 7.8 ° ± 0.2), 9.1 ° ± 0.5 (e.g., 9.1 ° ± 0.2), 11.5 ° ± 0.5 (e.g., 11.5 ° ± 0.2), 15.3 ° ± 0.5 (e.g., 15.3 ° ± 0.2), 16.0 ° ± 0.5 (e.g., 16.0 ° ± 0.2), 20.1 ° ± 0.5 (e.g., 20.1 ° ± 0.2), 25.4 ° ± 0.5 (e.g., 25.4 ° ± 0.2), and 26.6 ° ± 0.5 (e.6 ° ± 0.2) as measured by or calculated from X-ray diffraction by X-ray diffraction method with Cu ka X-ray. In this crystalline form of the substantially pure material, peaks can be observed at the angle of refraction 2θ as described in table 3. Alternatively or in addition, the crystalline form is characterized by a DSC profile showing five endothermic peaks at 58.3 ℃, 101.8 ℃, 129.8 ℃, 156.5 ℃ and 168.3 ℃.

TABLE 3 Table 3

Position [2 theta ]](+ -0.5, e.g. + -0.2)	Relative intensity
		5.7	48.91
7.8	100.00
		9.1	59.49
10.4	8.72
		11.5	24.53
12.9	8.50
		14.8	9.24
15.3	12.53
		16.0	14.09
17.2	7.22
		18.2	4.25
19.2	5.78
		20.1	14.54
21.5	6.47
		22.9	6.85
23.7	4.80
		25.4	65.68
26.6	14.53
		27.4	8.39
31.5	3.74
		34.2	4.36

In some embodiments, the crystalline form of the eplerin free base is crystalline form D of the eplerin free base and has peaks at diffraction angles 2θ (°) of 8.9 ° ± 0.5 (e.g., 8.9 ° ± 0.2), 10.3 ° ± 0.5 (e.g., 10.3 ° ± 0.2), and 26.0 ° ± 0.5 (e.g., 26.0 ° ± 0.2), as measured by or calculated from X-ray diffraction with Cu ka X-ray irradiation. In some embodiments, crystalline form D of the seppterin free base has peaks at diffraction angles θ (°) of 8.9 ° ± 0.5 (e.g., 8.9 ° ± 0.2), 10.3 ° ± 0.5 (e.g., 10.3 ° ± 0.2), 10.9 ° ± 0.5 (e.g., 10.9 ° ± 0.2), 17.8 ° ± 0.5 (e.g., 17.8 ° ± 0.2), 24.9 ° ± 0.5 (e.g., 24.9 ° ± 0.2), 26.0 ° ± 0.5 (e.g., 26.0 ° ± 0.2), 26.7 ° ± 0.5 (e.g., 26.7 ° ± 0.2), 26.8 ° ± 0.5 (e.g., 26.8 ° ± 0.2), and 28.3 ° ± 0.5 (e.g., 28.3 ° ± 0.2) as measured by or calculated from X-ray diffraction by X-ray diffraction method with Cu ka X-ray radiation. In this crystalline form of the substantially pure material, peaks can be observed at the angle of refraction 2θ as described in table 4. Alternatively or in addition, the crystalline form is characterized by a DSC profile showing three endotherms at 42.7 ℃, 66.3 ℃ and 232.9 ℃.

TABLE 4 Table 4

In some embodiments, the crystalline form of the eplerin free base is crystalline form a of the eplerin free base and has peaks at diffraction angles 2θ (°) of 4.7 ° ± 0.5 (e.g., 4.7 ° ± 0.2), 7.4 ° ± 0.5 (e.g., 7.4 ° ± 0.2), and 26.2 ° ± 0.5 (e.g., 26.2 ° ± 0.2), as measured by or calculated from X-ray diffraction by irradiation with Cu ka X-rays. In some embodiments, crystalline form a of the seppterin free base has peaks at diffraction angles 2 (°) of 4.7 ° ± 0.5 (e.g., 4.7 ° ± 0.2), 7.4 ° ± 0.5 (e.g., 7.4 ° ± 0.2), 9.5 ° ± 0.5 (e.g., 9.5 ° ± 0.2), 11.3 ° ± 0.5 (e.g., 11.3 ° ± 0.2), 15.6 ° ± 0.5 (e.g., 15.6 ° ± 0.2), 16.4 ° ± 0.5 (e.g., 16.4 ° ± 0.2), 26.2 ° ± 0.5 (e.g., 26.2 ° ± 0.2), and 27.2 ° ± 0.5 (e.g., 27.2 ° ± 0.2), as measured by or calculated from an X-ray diffraction method by irradiation with Cu ka X-ray. In this crystalline form of the substantially pure material, peaks can be observed at the angle of refraction 2θ as described in table 5. Alternatively or in addition, the crystalline form is characterized by a DSC profile showing endothermic peaks at 82.8 ℃ and 179.8 ℃.

TABLE 5

In some embodiments, the crystalline form of the eplerin free base is crystalline form E of the eplerin free base and has peaks at diffraction angles 2θ (°) of 6.0 ° ± 0.5, 6.0 ° ± 0.2, 10.6 ° ± 0.5, 10.6 ° ± 0.2, 12.1 ° ± 0.5 (e.g., 12.1 ° ± 0.2), 15.9 ° ± 0.5 (e.g., 15.9 ° ± 0.2), 20.9 ° ± 0.5 (e.g., 20.9 ° ± 0.2), and 24.6 ° ± 0.5 (e.g., 24.6 ° ± 0.2) as measured by or calculated from X-ray diffraction by X-ray diffraction with Cu ka X-ray. In some embodiments, crystalline form E of the seppterin free base has an angle of diffraction of, for example, 6.0 ° ± 0.5 (e.g., 6.0 ° ± 0.2), 10.6 ° ± 0.5 (e.g., 10.6 ° ± 0.2), 12.1 ° ± 0.5 (e.g., 12.1 ° ± 0.2), 15.9 ° ± 0.5 (e.g., 15.9 ° ± 0.2), 18.1 ° ± 0.5 (e.g., 18.1 ° ± 0.2), 20.9 ° ± 0.5 (e.g., 20.9 ° ± 0.2), 22.1 ° ± 0.5 (e.g., 22.1 ° ± 0.2), 24.6 ° ± 0.5 (e.g., 24.6 ° ± 0.2), 26.1 ° ± 0.5 (e.g., 26.1 ° ± 0.2), 28.1 ° ± 0.5 (e.g., 28.1 ° ± 0.2), 28.1 ° ± 0.5 (e.g., 28.2), 28.9 ° ± 0.5 (e.2) (e.g., 20.1 ° ± 0.2), and 32 ° -0.37 ° (e.2.2) (e.g., 32 °) at a diffraction angle of, e.g., 0.2.2.2 °). In a substantially pure form of this crystalline form, peaks can be observed at the angle of refraction 2θ as described in table 6. Alternatively or in addition, the crystalline form is characterized by a DSC profile showing two endothermic peaks at 112.9 ℃ and 195.8 ℃.

TABLE 6

In some embodiments, the crystalline form of the eplerin free base is crystalline form G of the eplerin free base and has peaks at diffraction angles 2θ (°) of 10.0 ° ± 0.5 (e.g., 10.0 ° ± 0.2), 10.6 ° ± 0.5 (e.g., 10.6 ° ± 0.2), and 25.7 ° ± 0.5 (e.g., 25.7 ° ± 0.2), as measured by or calculated from X-ray diffraction with Cu ka X-ray irradiation. In some embodiments, form G of the seppterin free base has diffraction peaks (°) at 10.0 ° ± 0.5 (e.g., 10.0 ° ± 0.2), 10.6 ° ± 0.5 (e.g., 10.6 ° ± 0.2), 11.2 ° ± 0.5 (e.g., 11.2 ° ± 0.2), 15.3 ° ± 0.5 (e.g., 15.3 ° ± 0.2), 15.9 ° ± 0.5 (e.g., 15.9 ° ± 0.2), 22.8 ° ± 0.5 (e.g., 22.8 ° ± 0.2), 24.4 ° ± 0.5 (e.g., 24.4 ° ± 0.2), 25.0 ° ± 0.5 (e.g., 25.0 ° ± 0.2), 25.7 ° ± 0.5 (e.g., 25.7 ° ± 0.2), and 26.6 ° ± 0.5 (e.6 ° ± 0.2) as measured by or calculated from X-ray diffraction by X-ray diffraction method irradiated with Cu ka X-ray. In this crystalline form of the substantially pure material, peaks can be observed at the angle of refraction 2θ as described in table 7.

TABLE 7

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In some embodiments, the crystalline form of the aminopterin hydrochloride has peaks at diffraction angles 2θ (°) of 7.8 ° ± 0.5 (e.g., 7.8 ° ± 0.2), 12.9 ° ± 0.5 (e.g., 12.9 ° ± 0.2), and 26.2 ° ± 0.5 (e.g., 26.2 ° ± 0.2), as measured by or calculated from X-ray diffraction with Cu ka X-ray irradiation. In some embodiments, the strongest peak in the X-ray diffraction pattern of the crystalline form of the aminopterin hydrochloride is observed at an angle of refraction 2 theta of 7.8 ° ± 0.5 (e.g., 7.8 ° ± 0.2). In a substantially pure material of this crystalline hydrochloride salt of aminopterin, peaks can be observed at the angle of refraction 2θ as described in table 8. Alternatively or in addition, the crystalline hydrochloride salt of aminopterin is characterized by a DSC curve showing an endotherm at 225.9 ℃.

TABLE 8

Position [2 theta ]](+ -0.5, e.g. + -0.2)	Relative intensity
		7.8	100.00
8.9	6.89
		12.9	58.56
15.6	8.52
		17.9	25.23
19.2	5.48
		21.1	10.97
23.6	25.15
		25.2	22.66
26.2	45.91
		27.6	32.94
30.3	10.50
		31.7	7.83
34.2	8.87
		36.7	3.67

In some embodiments, crystalline form 1 mesylate of methotrexate has peaks at diffraction angles 2θ (°) of 7.8 ° ± 0.5 (e.g., 7.8 ° ± 0.2), 23.5 ° ± 0.5 (e.g., 23.5 ° ± 0.2), and 29.0 ° ± 0.5 (e.g., 29.0 ° ± 0.2), as measured by or calculated from X-ray diffraction by Cu ka X-ray irradiation. In some embodiments, the strongest peak in the X-ray diffraction pattern is observed at an angle of refraction 2θ of 23.5°±0.5 (e.g., 23.5°±0.2). In a substantially pure material of crystalline form 1 mesylate salt of aminopterin, peaks can be observed at the angle of refraction 2θ as described in table 9. Alternatively or in addition, the crystalline form 1 mesylate salt of methotrexate is characterized by a DSC profile showing two endotherms at 186.0 ℃ and 229.1 ℃.

TABLE 9

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In some embodiments, the crystalline form 2 mesylate salt of methotrexate has peaks at diffraction angles 2θ (°) of 7.9 ° ± 0.5 (e.g., 7.9 ° ± 0.2), 23.4 ° ± 0.5 (e.g., 23.4 ° ± 0.2), and 28.9 ° ± 0.5 (e.g., 28.9 ° ± 0.2), as measured by or calculated from X-ray diffraction by Cu ka X-ray irradiation. In some embodiments, the strongest peak in the X-ray diffraction pattern is observed at an angle of refraction 2θ of 7.9°±0.5 (e.g., 7.9°±0.2). In a substantially pure material of crystalline form 2 mesylate salt of aminopterin, peaks can be observed at the angle of refraction 2θ as described in table 10. Alternatively or in addition, the crystalline form 2 mesylate salt of methotrexate is characterized by DSC curves showing three endotherms at 75.5 ℃, 182.6 ℃ and 234.9 ℃.

Table 10

Position [2 theta ]](+ -0.5, e.g. + -0.2)	Relative intensity
		7.9	100.00
11.0	21.32
		12.1	22.02
13.5	79.87
		15.7	11.87
17.8	9.81
		19.7	10.93
21.3	26.79
		23.4	96.13
24.1	24.88
		24.3	22.10
25.5	9.45
		26.0	11.27
27.6	7.63
		28.9	95.64
31.2	4.39
		36.1	6.65

In some embodiments, the crystalline form 3 mesylate salt of methotrexate has peaks at diffraction angles 2θ (°) of 21.7 ° ± 0.5 (e.g., 21.7 ° ± 0.2), 26.1 ° ± 0.5 (e.g., 26.1 ° ± 0.2), and 28.9 ° ± 0.5 (e.g., 28.9 ° ± 0.2), as measured by or calculated from X-ray diffraction by Cu ka X-ray irradiation. In some embodiments, the strongest peak in the X-ray diffraction pattern is observed at an angle of refraction 2θ of 26.1°±0.5 (e.g., 26.1°±0.2). In a substantially pure material of crystalline form 3 mesylate salt of aminopterin, peaks can be observed at the angle of refraction 2θ as described in table 11. Alternatively or in addition, the crystalline form 3 mesylate salt of methotrexate is characterized by a DSC profile showing two endotherms at 195.1 ℃ and 240.1 ℃.

TABLE 11

Position [2 theta ]](+ -0.5, e.g. + -0.2)	Relative intensity
		8.2	47.29
10.8	56.14
		12.6	16.34
13.2	15.90
		14.0	24.39
15.0	12.03
		15.9	16.20
18.2	22.97
		20.1	25.53
20.5	14.97
		21.3	22.70
21.7	71.48
		22.2	11.40
23.6	46.37
		24.8	44.00
25.5	9.08
		26.1	100.00
27.3	3.52
		28.9	68.42
31.2	4.49
		32.1	6.48
34.8	5.95
		35.6	1.67
39.1	2.91

In some embodiments, the crystalline nicotinate of the aminopterin has peaks at diffraction angles 2θ (°) of 9.5 ° ± 0.5 (e.g., 9.5 ° ± 0.2), 9.9 ° ± 0.5 (e.g., 9.9 ° ± 0.2), and 24.5 ° ± 0.5 (e.g., 24.5 ° ± 0.2), as measured by or calculated from X-ray diffraction with Cu ka X-ray irradiation. In some embodiments, the strongest peak in the X-ray diffraction pattern is observed at an angle of refraction 2θ of 24.5°±0.5 (e.g., 24.5°±0.2). In a substantially pure material of crystalline nicotinate of aminopterin, peaks can be observed at the angle of refraction 2θ as described in table 12. Alternatively or in addition, the crystalline nicotinate of aminopterin is characterized by a DSC profile showing an endotherm at 221.9 ℃.

Table 12

Position [2 theta ]](+ -0.5, e.g. + -0.2)	Relative intensity
		9.5	10.29
9.9	53.95
		11.5	9.31
12.0	11.76
		14.7	14.20
15.9	17.61
		17.5	7.53
19.0	5.37
		20.8	5.88
21.3	6.12
		21.7	7.20
23.2	34.05
		24.5	100.00
25.2	12.90
		28.0	8.51
31.1	5.39
		32.3	4.52
33.4	8.02
		35.1	5.05

In some embodiments, the crystalline p-toluenesulfonate salt has peaks at diffraction angles 2θ (°) of 6.5 ° ± 0.5 (e.g., 6.5 ° ± 0.2), 15.1 ° ± 0.5 (e.g., 15.1 ° ± 0.2), and 23.4 ° ± 0.5 (e.g., 23.4 ° ± 0.2) as measured by or calculated from X-ray diffraction with Cu ka X-ray irradiation. In some embodiments, the strongest peak in the X-ray diffraction pattern is observed at an angle of refraction 2θ of 6.5°±0.5 (e.g., 6.5°±0.2). In the substantially pure material of p-toluene sulfonate of aminopterin, peaks were observed at the refraction angles 2θ as described in table 13. Alternatively or in addition, the crystalline p-toluenesulfonate salt of aminopterin is characterized by a DSC curve showing three endotherms at 77.2 ℃, 202.4 ℃ and 260.2 ℃.

TABLE 13

Position [2 theta ]](+ -0.5, e.g. + -0.2)	Relative intensity
		6.5	100.00
12.9	1.79
		14.3	1.39
15.1	15.36
		16.2	5.33
18.4	8.96
		19.6	3.06
20.2	4.86
		21.8	2.23
22.5	2.95
		23.1	7.99
23.4	9.14
		24.5	1.81
26.0	2.48
		27.0	4.49
27.3	3.93
		28.1	5.31
28.4	5.59
		28.8	2.05
30.6	2.24
		31.0	1.98
32.6	1.82

In some embodiments, the crystalline benzenesulfonate salt of aminopterin has peaks at diffraction angles 2θ (°) of 6.5 ° ± 0.5 (e.g., 6.5 ° ± 0.2), 14.8 ° ± 0.5 (e.g., 14.8 ° ± 0.2), and 19.6 ° ± 0.5 (e.g., 19.6 ° ± 0.2), as measured by or calculated from X-ray diffraction with Cu ka X-ray radiation. In some embodiments, the strongest peak in the X-ray diffraction pattern is observed at an angle of refraction 2θ of 6.5°±0.5 (e.g., 6.5°±0.2). In a substantially pure material of the benzenesulfonate salt of aminopterin, peaks were observed at the angle of refraction 2θ as described in table 14. Alternatively or in addition, the crystalline benzenesulfonate salt of aminopterin is characterized by a DSC curve showing two endotherms at 202.3 ℃ and 265.5 ℃.

TABLE 14

Position [2 theta ]](+ -0.5, e.g. + -0.2)	Relative intensity
		4.9	5.90
6.5	100.00
		14.8	16.73
17.8	4.23
		19.6	7.98
21.5	2.49
		23.7	3.46
24.5	3.84
		26.1	3.29

In some embodiments, the crystalline phosphate of aminopterin has peaks at diffraction angles 2θ (°) of 16.6 ° ± 0.5 (e.g., 16.6 ° ± 0.2), 22.2 ° ± 0.5 (e.g., 22.2 ° ± 0.2), and 25.6 ° ± 0.5 (e.g., 25.6 ° ± 0.2), as measured by or calculated from X-ray diffraction with Cu ka X-ray irradiation. In some embodiments, the strongest peak in the X-ray diffraction pattern is observed at an angle of refraction 2θ of 25.6°±0.5 (e.g., 25.6°±0.2). In a substantially pure material of crystalline phosphate of aminopterin, peaks can be observed at the angle of refraction 2θ as described in table 15. Alternatively or in addition, the crystalline phosphate salt of aminopterin is characterized by a DSC profile showing three endotherms at 125.9 ℃, 152.1 ℃ and 157.6 ℃.

TABLE 15

In some embodiments, the crystalline malonate of aminopterin has peaks at diffraction angles 2θ (°) of 6.9 ° ± 0.5 (e.g., 6.9 ° ± 0.2), 22.7 ° ± 0.5 (e.g., 22.7 ° ± 0.2), and 23.8 ° ± 0.5 (e.g., 23.8 ° ± 0.2), as measured by or calculated from X-ray diffraction by Cu ka X-ray irradiation. In some embodiments, the strongest peak in the X-ray diffraction pattern is observed at an angle of refraction 2θ of 6.9°±0.5 (e.g., 6.9°±0.2). In a substantially pure material of crystalline malonate of aminopterin, peaks were observed at the angle of refraction 2θ as described in table 16. Alternatively or in addition, the crystalline malonate of the aminopterin is characterized by a DSC profile showing a melting event at 115.8 ℃.

Table 16

In some embodiments, the crystalline L-tartrate salt of aminopterin has peaks at diffraction angles 2θ (°) of 7.4 ° ± 0.5 (e.g., 7.4 ° ± 0.2), 14.2 ° ± 0.5 (e.g., 14.2 ° ± 0.2), and 21.8 ° ± 0.5 (e.g., 21.8 ° ± 0.2), as measured by or calculated from X-ray diffraction with Cu ka X-ray radiation. In some embodiments, the strongest peak in the X-ray diffraction pattern is observed at an angle of refraction 2θ of 7.4°±0.5 (e.g., 7.4°±0.2). In a substantially pure material of crystalline L-tartrate salt of aminopterin, peaks were observed at the refraction angles 2θ as described in table 17. Alternatively or in addition, the crystalline L-tartrate salt of methotrexate is characterized by DSC curves showing two endotherms at 97.2 ℃ and 160.6 ℃.

TABLE 17

In some embodiments, the crystalline gentisate of aminopterin has peaks at diffraction angles 2θ (°) of 7.1 ° ± 0.5 (e.g., 7.1 ° ± 0.2), 8.7 ° ± 0.5 (e.g., 8.7 ° ± 0.2) and 26.7 ° ± 0.5 (e.g., 26.7 ° ± 0.2) as measured by or calculated from X-ray diffraction by Cu ka X-ray irradiation. In some embodiments, the strongest peak in the X-ray diffraction pattern is observed at an angle of refraction 2θ of 7.1°±0.5 (e.g., 7.1°±0.2). In a substantially pure material of crystalline gentisate of aminopterin, peaks can be observed at the angle of refraction 2θ as described in table 18. Alternatively or in addition, the crystalline gentisate of aminopterin is characterized by a DSC profile showing three endotherms at 70.5 ℃, 128.2 ℃ and 184.7 ℃.

TABLE 18

In some embodiments, the crystalline fumarate salt of aminopterin has peaks at diffraction angles 2θ (°) of 11.4 ° ± 0.5 (e.g., 11.4 ° ± 0.2), 24.0 ° ± 0.5 (e.g., 24.0 ° ± 0.2), and 28.2 ° ± 0.5 (e.g., 28.2 ° ± 0.2), as measured by or calculated from X-ray diffraction by Cu ka X-ray irradiation. In some embodiments, the strongest peak in the X-ray diffraction pattern is observed at an angle of refraction 2θ of at least 24.0°±0.5 (e.g., 24.0°±0.2). In a substantially pure material of crystalline fumarate salt of aminopterin, peaks were observed at the angle of refraction 2θ as described in table 19. Alternatively or in addition, the crystalline fumarate salt of aminopterin is characterized by a DSC profile showing two endotherms at 114.3 ℃ and 229.7 ℃.

TABLE 19

In some embodiments, the crystalline glycolate has peaks at diffraction angles 2θ (°) of 7.6 ° ± 0.5 (e.g., 7.6 ° ± 0.2), 10.7 ° ± 0.5 (e.g., 10.7 ° ± 0.2), and 24.0 ° ± 0.5 (e.g., 24.0 ° ± 0.2) as measured by or calculated from X-ray diffraction with Cu ka X-ray irradiation. In some embodiments, the strongest peak in the X-ray diffraction pattern is observed at an angle of refraction 2θ of at least 7.6°±0.5 (e.g., 7.6°±0.2). In a substantially pure material of crystalline glycolate of aminopterin, peaks can be observed at the refraction angles 2θ as described in table 20. Alternatively or in addition, the crystalline glycolate of aminopterin is characterized by a DSC profile showing two endotherms at 133.9 ℃ and 147.7 ℃.

Table 20

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In some embodiments, the crystalline acetate has peaks at diffraction angles 2θ (°) of 6.2 ° ± 0.5 (e.g., 6.2 ° ± 0.2), 12.0 ° ± 0.5 (e.g., 12.0 ° ± 0.2), and 18.1 ° ± 0.5 (e.g., 18.1 ° ± 0.2), as measured by or calculated from X-ray diffraction with Cu ka X-ray irradiation. In some embodiments, the strongest peak in the X-ray diffraction pattern is observed at an angle of refraction 2θ of at least 6.2°±0.5 (e.g., 6.2°±0.2). In a substantially pure material of crystalline acetate salt of aminopterin, peaks can be observed at the angle of refraction 2θ as described in table 21. Alternatively or in addition, the crystalline acetate salt of aminopterin is characterized by a DSC profile showing two endotherms at 146.1 ℃ and 175.4 ℃.

Table 21

In some embodiments, crystalline form 1 sulfate of aminopterin has peaks at diffraction angles 2θ (°) of 5.1 ° ± 0.5 (e.g., 5.1 ° ± 0.2), 7.8 ° ± 0.5 (e.g., 7.8 ° ± 0.2), and 23.0 ° ± 0.5 (e.g., 23.0 ° ± 0.2), as measured by or calculated from X-ray diffraction with Cu ka X-ray radiation. In some embodiments, the strongest peak in the X-ray diffraction pattern is observed at an angle of refraction 2θ of 5.1°±0.5 (e.g., 5.1°±0.2). In a substantially pure material of crystalline form 1 sulfate salt of aminopterin, peaks can be observed at the angle of refraction 2θ as described in table 22. Alternatively or in addition, crystalline form 1 sulfate salt of aminopterin is characterized by a DSC profile showing three endotherms at 94.5 ℃, 158.3 ℃ and 209.9 ℃.

Table 22

In some embodiments, crystalline form 2 sulfate of aminopterin has peaks at diffraction angles 2θ (°) of 7.8 ° ± 0.5 (e.g., 7.8 ° ± 0.2), 8.8 ° ± 0.5 (e.g., 8.8 ° ± 0.2), and 24.1 ° ± 0.5 (e.g., 24.1 ° ± 0.2), as measured by or calculated from X-ray diffraction with Cu ka X-ray irradiation. In some embodiments, the strongest peak in the X-ray diffraction pattern is observed at an angle of refraction 2θ of 8.8°±0.5 (e.g., 8.8°±0.2). In a substantially pure material of crystalline form 2 sulfate salt of aminopterin, peaks can be observed at the angle of refraction 2θ as described in table 23.

Table 23

The invention may employ a pharmaceutical composition comprising a pharmaceutically acceptable excipient and an effective amount of methotrexate or a pharmaceutically acceptable salt thereof. Examples of pharmaceutical compositions of aminopterin and salts thereof can be found in WO 2019/046849 and WO 2019/232120, the compositions of which are incorporated herein by reference in their entirety.

The pharmaceutically acceptable excipients may be any of those conventionally used and are limited only by chemo-physical considerations such as solubility and route of administration. Those skilled in the art will appreciate that, in addition to the pharmaceutical compositions described below, the aminopterin may be formulated as an inclusion complex or liposome, such as a cyclodextrin inclusion complex.

Pharmaceutically acceptable excipients (e.g., vehicles, adjuvants, excipients, or diluents) described herein are well known to those skilled in the art and are readily available to the public. Preferably, the pharmaceutically acceptable excipients are excipients that are chemically inert to the aminopterin and excipients that have no deleterious side effects or toxicity under the conditions of use.

Formulations for increasing gastric and/or foregut residence time

Gastric retentive drug delivery is a method in which the drug formulation is designed to stay in the stomach longer (e.g., until drug release is completed).

Bioadhesive dosage forms utilize polymers that adhere to surfaces and result in controlled release of the drug. The bioadhesive polymer may be anionic (e.g., carboxymethyl cellulose, alginic acid, polyacrylic acid, pectin, carrageenan, polycarbophil (polycarbophil) or carbomer); cationic (e.g., chitosan, polylysine, or polybrene); or nonionic (e.g., polyethylene glycol, polyvinylpyrrolidone, dextran, or hydroxypropyl methylcellulose).

The high density dosage forms are designed to be located in the stomach at a level below the pyloric sphincter and thereby avoid emptying. Excipients suitable for high density dosage forms include iron powder, barium sulfate, zinc oxide and titanium oxide.

The expandable dosage form is designed to expand in the stomach to be greater than the pyloric sphincter and thereby avoid emptying. For example, dosage forms comprising a drug core, a swellable hydrocolloid, and an outer semipermeable polymer are suitable for expandable dosage forms.

Similar to the swellable dosage form, the superporous hydrogel dosage form is designed to swell in the stomach to be greater than the pyloric sphincter. The superporous hydrogel dosage form can include a polymer, such as croscarmellose sodium.

The floating dosage form is designed to have a density lower than gastric juice. The floating dosage form may comprise a composition comprising an ion exchange resin, a raft system, a plenum, an effervescent mixture, a swellable hydrocolloid, or a multiparticulate system.

Antioxidant agent

The aminopterin is prone to rapid oxidation upon exposure to air. Accordingly, the pharmaceutical composition of the present invention may include an antioxidant. Antioxidants can minimize oxidative degradation of the aminopterin. Examples of antioxidants include, but are not limited to, 4-chloro-2, 6-di-tert-butylphenol, tocopherol, alpha-tocopherol, alkylated diphenylamine, ascorbic acid, myristate, palmitate, stearate, beta-carotene, butylated hydroxyanisole, butylated hydroxytoluene, citric acid, cysteine, D-alpha-tocopheryl polyethylene glycol 1000 succinate, desferrioxamine mesylate, dodecyl gallate, ethylenediamine tetraacetic acid, ethyl p-hydroxybenzoate, folic acid, fumaric acid, gallic acid, glutathione, lecithin, malic acid, methyl p-hydroxybenzoate, monothioglycerol, N-acetylcysteine, nordihydroguaiacic acid, octyl gallate, p-phenylenediamine, potassium ascorbate, potassium metabisulfite, potassium sorbate, propionic acid, propyl gallate, retinol, sorbic acid, sodium ascorbate, sodium bisulphite, sodium metabisulfite, sodium thiosulfate, tartaric acid, tertiary butylhydroquinone, tocopheryl acetate, vitamin A, vitamin B6, vitamin B, vitamin E, or vitamin E. Examples of antioxidants include, but are not limited to, ascorbic acid, tocopherol, retinol, palmitic acid ascorbyl ester, N-acetylcysteine, glutathione, ethylenediamine tetraacetic acid, sodium bisulfite, sodium metabisulfite, thiourea, butylated hydroxytoluene, butylated hydroxyanisole, and vitamin E. In some embodiments, the pharmaceutical compositions of the present invention include ascorbic acid, tocopherol, retinol, palmitoleic acid ascorbate, N-acetylcysteine, glutathione, butylated hydroxytoluene, and/or butylated hydroxyanisole as antioxidants.

In some embodiments, the pharmaceutical composition comprises less than 10% by weight of antioxidants, e.g., less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% or substantially no antioxidants. In some embodiments, the pharmaceutical composition comprises 2-9% antioxidant, such as 2-4%, 3-5%, 4-6%, 5-7%, 6-8% or 7-9% by total weight. In some embodiments, the pharmaceutical composition comprises 5-100% of the highest daily dose of USP of the antioxidant, e.g., in some embodiments, the pharmaceutical composition comprises about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the highest daily dose of USP of the antioxidant. In some embodiments, the ratio of the aminopterin to the antioxidant is at least 1:1, e.g., 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1 by weight.

Dispersing agent

In some embodiments, the pharmaceutical compositions of the present invention comprise at least one dispersing agent. The dispersing agent may separate particles in the formulation, for example, releasing its pharmaceutical substance upon contact with moisture. Examples of dispersants include, but are not limited to, crosslinked polyvinylpyrrolidone, carboxymethyl cellulose (e.g., a salt of crosslinked carboxymethyl cellulose, such as sodium crosslinked carboxymethyl cellulose), starch (e.g., sodium starch glycolate), or alginic acid. In some embodiments, the dispersing agent in the pharmaceutical composition is carboxymethyl cellulose, such as a pharmaceutically acceptable salt of croscarmellose. In some embodiments, the pharmaceutical composition may include 0.1-1.5% dispersant, such as about 0.1%, 0.5%, 1% or 1.5% by total weight. In some embodiments, the pharmaceutical composition comprises less than 1.5% dispersant, such as less than 1%, less than 0.5%, or less than 0.1% by total weight.

Anti-caking agent

In some embodiments, the pharmaceutical compositions of the present invention comprise at least one anti-caking agent. In some embodiments, the pharmaceutical composition comprises at least two anti-caking agents. Exemplary anti-caking agents include colloidal silica, microcrystalline cellulose, tricalcium phosphate, microcrystalline cellulose, magnesium stearate, sodium bicarbonate, sodium ferrocyanide, potassium ferrocyanide, calcium phosphate, sodium silicate, colloidal silica, calcium silicate, magnesium trisilicate, talc, sodium aluminosilicate, potassium aluminum silicate, calcium aluminosilicate, bentonite, aluminum silicate, stearic acid, and polydimethylsiloxane. In some embodiments, the at least one anti-caking agent is colloidal silica or microcrystalline cellulose. In some embodiments, the pharmaceutical composition may include 65-75% of the anti-caking agent, such as about 65%, 67%, 70%, 73%, or 75% by total weight. In some embodiments, the pharmaceutical composition includes both colloidal silica and microcrystalline cellulose. In some embodiments, the pharmaceutical composition comprises 60-65% microcrystalline cellulose by total weight and 5-7% colloidal silicon dioxide by total weight.

Administration vehicle

In some embodiments, the pharmaceutical compositions of the present invention are combined with an administration vehicle prior to administration, e.g., an administration vehicle having a viscosity of about 50-1750 centipoise (cP). One type of suspending agent that may be used is a combination of glycerin and sucrose in water (e.g., having 2.5% glycerin and 27% sucrose in water)Oral mixture). An appropriate amount of the composition may be added to the administration vehicle mixture immediately prior to administration and agitated to suspend the composition.

Other suspending agents may also be used as delivery vehicles. Exemplary suspending agents include water, agar, alginic acid, sodium carboxymethyl cellulose, carrageenan, dextrin, gelatin, guar gum, hydroxyethyl cellulose, hydroxypropyl methylcellulose, methyl cellulose, polyethylene glycol, povidone, tragacanth, xanthan gum, or other suspending agents known in the art.

Dosage of

The aminopterin or pharmaceutically acceptable salt thereof may be used in any suitable dosage. Suitable dosages and dosage regimens may be determined by conventional scoping techniques. Typically, treatment is initiated at a smaller dose than the optimal dose. Thereafter, the dose is increased in small increments until the optimal effect in this case is reached. For convenience, the total daily dose may be divided and administered in portions throughout the day, if desired. The present invention provides a broad range of responses with appropriate dosages and appropriate administration of certain compounds. Typically, the dosage will range from about 1 to about 150mg/kg or from about 2.5 to about 150mg/kg of body weight of the subject being treated per day, for example 60 mg/kg/day. For example, in embodiments, about 10mg/kg to about 150mg/kg, about 20mg/kg to about 150mg/kg, about 10mg/kg to about 60mg/kg, about 20mg/kg to about 60mg/kg, about 40mg/kg to about 100mg/kg, about 100mg/kg to about 150mg/kg, about 60mg/kg to about 120mg/kg, about 80mg/kg to about 100mg/kg, about 40mg/kg to about 60mg/kg, about 2.5mg/kg to about 20mg/kg, about 2.5mg/kg to about 10mg/kg or about 2.5mg/kg to about 5mg/kg of subject body weight of the methotrexate, or pharmaceutically acceptable salts thereof, may be administered daily, one or more times per day to obtain the desired therapeutic effect.

In some embodiments, the aminopterin or pharmaceutically acceptable salt thereof may be formulated into a unit solid oral dosage form, such as a granule. In these embodiments, each unit solid oral dosage form (e.g., pouch (sachets)) may comprise any suitable amount of the aminopterin or pharmaceutically acceptable salt thereof. For example, each solid oral dosage form may comprise about 2.5mg, about 5mg, about 10mg, about 20mg, about 30mg, about 40mg, about 50mg, about 60mg, about 70mg, about 80mg, about 90mg, about 100mg, about 125mg, about 150mg, about 175mg, about 200mg, about 225mg, about 250mg, about 275mg, about 300mg, about 325mg, about 350mg, about 375mg, about 400mg, about 425mg, about 450mg, about 475mg, about 500mg, about 750mg, about 1g, about 1.25g, or about 1.5g.

The aminopterin or a pharmaceutically acceptable salt thereof may be used to prepare a liquid formulation, for example in the form of a solution, suspension or emulsion. Formulations suitable for oral administration include, but are not limited to, (a) capsules, sachets, tablets, lozenges and troches (troche), each containing a predetermined amount of the active ingredient as a solid or as granules; (b) a powder; (c) A liquid solution, such as an effective amount of the compound dissolved in a diluent such as water, saline, or orange juice; (d) suspensions in suitable liquids; and (e) a suitable emulsion. Preferred are solid oral dosage forms such as capsule form, tablet form and powder form. The capsule form may be of the conventional hard or soft shell gelatin type containing, for example, surfactants, lubricants and inert fillers such as lactose, sucrose, calcium phosphate and corn starch. Tablet forms may include one or more of the following: lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, gum acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid and other excipients, colorants, diluents, buffers, disintegrants, wetting agents, preservatives, flavoring agents and pharmacologically compatible excipients. Lozenge forms may comprise the active ingredient in a flavoring agent (typically sucrose and acacia or tragacanth), and pastilles (pastilles) comprise the active ingredient in an inert base (such as gelatin and glycerin, or sucrose and acacia), emulsions, gels, and the like containing, in addition to the active ingredient, excipients such as are known in the art.

Formulations suitable for oral and/or parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents, solubilising agents, thickening agents, stabilisers and preservatives. The compounds may be administered in pharmaceutical excipients such as sterile liquids or liquid mixtures, including aqueous saline, aqueous dextrose and related sugar solutions, alcohols such as ethanol, benzyl alcohol or cetyl alcohol, glycols such as propylene glycol or polyethylene glycol and other polyvinyl alcohols, glycerol ketals such as 2, 2-dimethyl-1, 3-dioxolane-4-methanol, ethers such as poly (ethylene glycol) 400, oils, fatty acid esters or glycerides, or acetylated fatty acid glycerides with or without the addition of pharmaceutically acceptable surfactants (such as soaps or detergents), suspending agents such as pectin, carbomers, methylcellulose, hydroxypropyl methylcellulose or carboxymethylcellulose, or emulsifiers and other pharmaceutical adjuvants.

The invention features a pharmaceutical composition in an orally tolerable formulation containing a therapeutically effective amount of aminopterin and less than 10% of an antioxidant. In some embodiments, the pharmaceutical composition is a particulate formulation dispersed in a pharmaceutically acceptable excipient, e.g., the composition may be mixed into water and ingested by the subject (e.g., over a period of 5 to 10 minutes). Suitable formulations for use in the present invention are found in Remington's Pharmaceutical Sciences, mack Publishing Company, philiadelphia, PA 22 th edition, 2010. Except insofar as any conventional excipient is incompatible with the active ingredient, its use in pharmaceutical compositions is contemplated. Furthermore, for animal (e.g., human) administration, it will be appreciated that the preparation should meet sterility, pyrogenicity, general safety and purity standards as required by the FDA biosystems office (FDA Office ofBiological Standards).

Solid dosage form for oral administration

Formulations for oral use include granules containing the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients, and such formulations are known to the skilled artisan (e.g., U.S. Pat. Nos. 5,817,307, 5,824,300, 5,830,456, 5,846,526, 5,882,640, 5,910,304, 6,036,949, 6,372,218, which are incorporated by reference). Excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches (including potato starch), calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives (including microcrystalline cellulose), starches (including potato starch), croscarmellose sodium, alginates, or alginic acid); binders (e.g. sucrose, glucose, sorbitol, gum arabic, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, polyvinylpyrrolidone or polyethylene glycol); and lubricants, glidants, anti-adherent agents (e.g., magnesium stearate, zinc stearate, stearic acid, silicon dioxide, hydrogenated vegetable oil, or talc), anti-caking agents (e.g., colloidal silicon dioxide, microcrystalline cellulose, tricalcium phosphate, microcrystalline cellulose, magnesium stearate, sodium bicarbonate, sodium ferrocyanide, potassium ferrocyanide, calcium phosphate, sodium silicate, colloidal silicon dioxide, calcium silicate, magnesium trisilicate, talc, sodium aluminosilicate, potassium aluminum silicate, calcium aluminosilicate, bentonite, aluminum silicate, stearic acid, polydimethylsiloxane). Other pharmaceutically acceptable excipients may be colorants, flavors, plasticizers, humectants and buffers. In some embodiments, an excipient (e.g., flavoring agent) is packaged with the composition. In some embodiments, the excipient (e.g., flavoring agent) is packaged separately from the composition (e.g., combined with the composition prior to administration).

The solid compositions of the present invention may include a coating suitable for protecting the composition from undesired chemical changes, such as chemical degradation prior to release of the active substance. The coating may be applied to the solid dosage form in a manner similar to that described in Encyclopedia ofPharmaceutical Technology (supra).

The powders and granules can be prepared in a conventional manner using the above-mentioned ingredients, using, for example, mixers, fluidized bed devices, melt coagulation devices, rotor granulator, extrusion/spheronizer or spray drying equipment.

Therapeutic method

The aminopterin or a pharmaceutically acceptable salt thereof serves as a useful therapeutic agent for the treatment of glioblastoma. In some embodiments of any one of the methods described herein, the method further comprises therapeutic radiation treatment. In some embodiments of any of the methods described herein, the method further comprises administering a chemotherapeutic agent (e.g., temozolomide). Thus, various forms of aminopterin or pharmaceutically acceptable salts thereof according to the present invention may be administered to a subject in an amount effective to obtain treatment or amelioration of a disease, disorder, or condition.

In some embodiments of any of the methods described herein, the method further comprises therapeutic radiation. In some embodiments of any of the methods described herein, the amount of radiation delivered during the treatment cycle may be at least about 1Gy, at least about 2Gy, at least about 5Gy, at least about 10Gy, at least about 20Gy, at least about 30Gy, at least about 40Gy, at least about 50Gy, at least about 60Gy, at least about 70Gy, at least about 80Gy, at least about 90Gy, at least about 100Gy, at least about 150Gy, at least about 200Gy, at least about 300Gy, at least about 400Gy, or at least about 500Gy. In various embodiments, the radiation dose is divided into more than one portion, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 or more portions, for each treatment cycle. In various embodiments, the treatment period may be at least about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, or at least about 10 weeks. In various embodiments, one or more treatment cycles (e.g., 2, 3, 4, 5, or more treatment cycles) may be utilized.

In some embodiments of any of the methods described herein, the method further comprises administering the additional therapeutic agent to the subject in need thereof. In some embodiments of any of the methods described herein, the methotrexate or pharmaceutically acceptable salt thereof is administered at least 1, 2, 3, 4, 5, 6, 7, 10 days or more prior to the therapeutic radiation.

In some embodiments of any of the methods described herein, the method further comprises administering the additional therapeutic agent to the subject in need thereof. In some embodiments of any of the methods described herein, the methotrexate or pharmaceutically acceptable salt thereof is administered at least 1, 2, 3, 4, 5, 6, 7, 10, 20, 30, 60, 90, 120, 150, 180, 210, 300 days or more after the therapeutic radiation.

In some embodiments of any one of the methods described herein, the method further comprises administering a chemotherapeutic agent. A non-limiting example of a chemotherapeutic agent is Temozolomide (TMZ). In various embodiments, the dose of TMZ is from about 1 to about 1000mg/m per day as measured by the surface area of the subject's body ² . In various embodiments, the TMZ dose is from about 1 to about 500mg/m per day ² Or about 1 to about 250mg/m per day ² Or about 1 to about 100mg/m per day ² Or about 10 to about 500mg/m per day ² Or about 50 to about 250mg/m per day ² Or about 50 to about 100mg/m per day ² . The chemotherapeutic agent (e.g., TMZ) may be administered for at least about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks. In various embodiments, the chemotherapeutic agent (e.g., TMZ) is administered prior to, concurrently with, and/or after administration of the aminopterin or pharmaceutically acceptable salt thereof. In various embodiments, the chemotherapeutic agent (e.g., TMZ) is administered prior to, concurrent with, and/or after the administration of the therapeutic radiation treatment. In addition, chemotherapeutic agents (e.g., TMZ) may be present during the maintenance periodA staging period, wherein the chemotherapeutic agent (e.g., TMZ) is administered for at least 1 day during, for example, a 28 day treatment period, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 days during a 28 day maintenance treatment period.

The methotrexate or pharmaceutically acceptable salts thereof may be administered to the subject before, during, and/or after therapeutic radiation and/or administration of the chemotherapeutic agent. In various embodiments of any of the methods described herein, the methotrexate or pharmaceutically acceptable salt thereof is administered for at least about 1, 2, 3, 5, 6, 10, 14, 21, 28, or 30 days prior to the commencement of radiation therapy. In various embodiments of any of the methods described herein, the methotrexate or pharmaceutically acceptable salt thereof is administered continuously (e.g., daily) throughout the radiation treatment. In various embodiments of any of the methods described herein, the method further comprises administering the methotrexate or pharmaceutically acceptable salt thereof for at least about 1, 2, 3, 5, 6, 10, 14, 21, 28, 30, 60, 90, 120, 150, 180, 210, 240, or more days after the radiation treatment. In various embodiments of any of the methods described herein, the methotrexate or pharmaceutically acceptable salt thereof is administered for at least about 1, 2, 3, 5, 6, 10, 14, 21, 28, or 30 days prior to initiation of the chemotherapeutic agent treatment (e.g., TMZ treatment). In various embodiments of any of the methods described herein, the methotrexate or pharmaceutically acceptable salt thereof is administered continuously (e.g., daily) throughout the chemotherapeutic agent treatment (e.g., TMZ treatment). In various embodiments of any of the methods described herein, the method comprises administering the methotrexate or pharmaceutically acceptable salt thereof for at least about 1, 2, 3, 5, 6, 10, 14, 21, 28, 30, 60, 90, 120, 150, 180, 210, 240, or more days after the chemotherapeutic agent treatment (e.g., TMZ treatment).

In some embodiments of any of the methods described herein, the subject has the following treatment schedule: (1) An induction period of only aminopterin or a pharmaceutically acceptable salt thereof (e.g., only aminopterin or a pharmaceutically acceptable salt thereof for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days); (2) Aminopterin or a pharmaceutically acceptable salt thereof + a chemotherapeutic agent (e.g., TMZ) + a radiation period of radiation (e.g., for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more weeks); (3) A chemotherapeutic "break" period (e.g., only aminopterin or a pharmaceutically acceptable salt thereof, no chemotherapeutic agent (e.g., TMZ)); (4) A maintenance period comprising one or more 28-day periods, e.g., 2, 3, 4, 5, 6, 7, 8 or more periods, wherein the aminopterin or pharmaceutically acceptable salt thereof is administered continuously throughout the period, but wherein the chemotherapeutic agent (e.g., TMZ) is administered only for the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days of each 28-day period.

In some embodiments, the subject is a child (e.g., the subject is less than 18 years old, less than 17 years old, less than 16 years old, less than 15 years old, less than 14 years old, less than 13 years old, less than 12 years old, less than 11 years old, less than 10 years old, less than 9 years old, less than 8 years old, less than 7 years old, less than 6 years old, less than 5 years old, less than 4 years old, less than 3 years old, less than 2 years old, less than 1 year old). In some embodiments, the subject is an adult (e.g., the subject is older than 18 years). In some embodiments, the subject is at least 20 years old, at least 30 years old, at least 40 years old, at least 50 years old, at least 60 years old, at least 70 years old, at least 80 years old.

The aminopterin or pharmaceutically acceptable salt thereof may be administered with or without food. Without being bound by theory, administration of the aminopterin with food results in BH, for example, by decreasing the rate of absorption of the aminopterin ₄ An increase in plasma exposure. If the administered aminopterin is rapidly absorbed, for example, by fasting administration, the cell's aminopterin reductase and/or dihydrofolate reductase may become saturated beyond V _max Resulting in at least a portion of the applied aminopterin being unreduced to 7, 8-dihydrobiopterin and subsequent BH ₄ Is left from the cell. These excess amounts of aminopterin may then be not converted to BH at all times ₄ Is excreted, resulting in BH in plasma compared to administration of aminopterin with food ₄ At lower levels, administration of the aminopterin with food decreases the rate of absorption of the aminopterin or increases the rate of absorption of the aminopterin and results in a V that has a rate of response that is lower, equal to, or slightly higher than the saturation of substrates of the aminopterin reductase and/or dihydrofolate reductase _max . Administration of aminopterin or a pharmaceutically acceptable salt thereof with food unexpectedly results in a maximum BH compared to administration without food ₄ Increased plasma concentration (Cmax) and exposure, e.g. by BH ₄ The area under the concentration time curve from zero time to the final concentrationAUC _0-last ) Measured. For example, an effective amount of aminopterin or a pharmaceutically acceptable salt thereof is sufficient to produce a BH of at least 50ng/mL (e.g., at least 60ng/mL, at least 100ng/mL, at least 200ng/mL, at least 400ng/mL, at least 600ng/mL, at least 1000ng/mL, or at least 2000ng/mL, or 50ng/mL to 100ng/mL, 60ng/mL to 400ng/mL, 200ng/mL to 600ng/mL, 400ng/mL to 1000ng/mL, or 600ng/mL to 1500 ng/mL) in the plasma of a subject within 10 hours of administration with a food ₄ Concentration amounts (e.g., 1.0mg/kg to 100mg/kg per dose or 2.5mg/kg to 100mg/kg per dose). An effective amount may comprise an amount sufficient to produce a maximum BH of at least 50ng/mL (e.g., at least 60ng/mL, at least 100ng/mL, at least 200ng/mL, at least 400ng/mL, at least 600ng/mL, at least 1000ng/mL, or at least 2000ng/mL, or 50ng/mL to 100ng/mL, 60ng/mL to 400ng/mL, 200ng/mL to 600ng/mL, 400ng/mL to 1000ng/mL, or 600ng/mL to 1500 ng/mL) in the plasma of a subject, than in 10 hours of administration of aminopterin or a pharmaceutically acceptable salt thereof without food ₄ The dose of plasma concentration (Cmax) is at least 5% (at least 10%, at least 20%, at least 50%, at least 70%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140% or at least 150%) lower than the dose.

In some embodiments of any of the methods described herein, the food is a high protein food. In some embodiments of any of the methods described herein, the food is a high fat food (e.g., at least 25, 30, 40, or 50% of the calories from fat). In some embodiments of any of the methods described herein, the food is a high protein and high fat food. In some embodiments, the food is a high calorie food (e.g., the food comprises at least 100 calories, such as at least 200 calories, at least 300 calories, at least 400 calories, at least 500 calories, such as 500-1500 or 800-1000 calories). In some embodiments of any of the methods described herein, the food is a meal, such as breakfast, lunch or dinner. The aminopterin or pharmaceutically acceptable salt thereof may be provided in a composition separate from the food being consumed (e.g., the aminopterin or pharmaceutically acceptable salt thereof is not incorporated into the food product). In some embodiments of any of the methods described herein, the food is a low fat food.

Administration to the subject may occur less than 30 minutes prior to consumption of the food or after consumption of the food, e.g., immediately prior to consumption of the food up to 1 hour after consumption, such as substantially simultaneous with the food. Administration with food (e.g., less than 30 minutes before eating food or after eating food, e.g., immediately before eating food up to 1 hour after eating) can result in BH compared to administration without food (e.g., more than 2 hours after eating food up to 30 minutes before eating other food) ₄ Cmax or BH of (C) ₄ And the extent of production of (c) and the resulting plasma exposure (AUC _0-last ) An increase (e.g., at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150%).

The methotrexate or pharmaceutically acceptable salt thereof may be administered to the subject in the absence of food, e.g., more than 30 minutes before eating the food or at least 2 hours after eating the food. In some embodiments of any of the methods described herein, the administering occurs more than 30 minutes prior to eating the food or at least 3 hours after eating the food. In some embodiments of any of the foregoing methods, the method further comprises administering the aminopterin or pharmaceutically acceptable salt thereof to the subject in the absence of a high protein food. In some embodiments of any of the foregoing methods, the methotrexate or pharmaceutically acceptable salt thereof is administered in the absence of high fat foods (e.g., at least 25, 30, 40, or 50% of calories from fat). In some embodiments of any of the foregoing methods, the method further comprises administering the aminopterin or pharmaceutically acceptable salt thereof in the absence of a high protein and high fat food. In some embodiments, the methotrexate or pharmaceutically acceptable salt thereof is administered without high calorie foods (e.g., foods comprising at least 100 calories, such as at least 200 calories, at least 300 calories, at least 400 calories, at least 500 calories, such as 500-1500 or 800-1000 calories). In some embodiments of any of the foregoing methods, the methotrexate or pharmaceutically acceptable salt thereof is administered without food as a meal (e.g., breakfast, lunch, or dinner).

Without being bound by theory, administration of the aminopterin or pharmaceutically acceptable salt thereof in the absence of food may result in increased plasma, CSF, and/or brain exposure of the aminopterin by increasing the rate of absorption of the aminopterin. Because the aminopterin passes through the cell membrane efficiently, if the administered aminopterin is rapidly absorbed, such as by fasting administration, the active transporter of the aminopterin and/or the aminopterin reductase in the cell may saturate, resulting in at least a portion of the administered aminopterin not entering the cell and/or leaving the cell without being reduced to 7, 8-dihydrobiopterin. These excess amounts of aminopterin in plasma can then be converted to BH ₄ Previously crossing the Blood Brain Barrier (BBB) and entering brain cells, resulting in BH in the brain (and/or CSF) compared to administration with food ₄ Higher levels, administration with food reduced the rate of uptake of the aminopterin and may not result in saturation of the aminopterin transporter and intracellular aminopterin reductase. Thus, administration of aminopterin or a pharmaceutically acceptable salt thereof in the absence of food unexpectedly results in a maximum plasma, CSF and/or brain concentration (Cmax) and/or extent of Absorption (AUC) of the aminopterin as compared to administration with food _0-last ) And (3) increasing. During treatment, increased levels of aminopterin in plasma, CSF, and/or brain may be beneficial.

The amount of the actual dosage of the composition of the invention administered to a subject may be determined by physical and physiological factors such as body weight, severity of the condition, type of disease being treated, previous or concurrent therapeutic intervention, idiopathic disease of the subject, and the route of administration. Depending on the dosage and route of administration, the preferred dosage and/or number of times an effective amount is administered may vary depending on the subject's response. In any event, the practitioner responsible for administration will determine the concentration of the active ingredient in the composition and the appropriate dosage for the individual subject.

In some embodiments, the subject receives about 1mg/kg to 120mg/kg (e.g., about 10mg/kg to about 60mg/kg, about 20mg/kg to about 60mg/kg, or about 20mg/kg, about 30mg/kg, about 40mg/kg, about 50mg/kg, about 60 mg/kg) per dose. The subject may receive a pharmaceutical composition comprising aminopterin or a pharmaceutically acceptable salt thereof once per day, twice per day, or three times per day during the treatment period. In some embodiments, the subject may not be allowed to take any drug known to inhibit folate synthesis (e.g., methotrexate (methotrexate), pemetrexed (pemetrexed), or trimellitate). The aminopterin or pharmaceutically acceptable salt thereof may be administered in two equal doses (e.g., two doses at different times of the day), such as two 60mg/kg doses (e.g., one 60mg/kg dose in the morning and one 60mg/kg dose in the evening), two 40mg/kg doses (e.g., one 40mg/kg dose in the morning and one 40mg/kg dose in the evening), two 30mg/kg doses (e.g., one 30mg/kg dose in the morning and one 30mg/kg dose in the evening), two 20mg/kg doses (e.g., one 20mg/kg dose in the morning and one 20mg/kg dose in the evening), or two 10mg/kg doses (e.g., one 10mg/kg dose in the morning and one 10mg/kg dose in the evening).

Equivalents and scope

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the invention is not intended to be limited to the above description, but rather is set forth in the following claims.

Furthermore, it will be understood that any particular embodiment of the invention falling within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are believed to be known to those skilled in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the present invention (e.g., any compound, any method of manufacture, any method of use, etc.) may be excluded from any one or more of the claims for any reason, whether or not related to the presence of the prior art.

Examples

Example 1.

On day 0, orthotopic glioblastomas (n=13/group) were established in nude mice (U87) and homologous (GL 261) mouse models. Oral administration of the aminopterin (SP) in the U87 mouse model was continued for 6 days (10 mg/kg of aminopterin) or daily (1 mg/kg of aminopterin) until the end of the experiment (60 days) starting on day 6. SP (10 mg/kg) was orally administered daily starting on day 6 in the GL261 mouse model until the end of the experiment (60 days). Radiation (IR) (2 Gy of 5 fxs) with 3D treatment planning and delivery starts on day 13 and ends on day 17.

The results from the U87 mice are shown in figure 1. As shown in fig. 1, administration of the aminopterin improved survival relative to control (i.e., no IR or SP) or IR alone as follows: both the 10mg/kg lasting 6 days+IR and the continuous 1mg/kg+IR groups differed significantly from the control group. The difference between the 10mg/kg 6-day lasting group and the IR group alone did not reach statistical significance. The consecutive 1mg/kg groups were significantly different from all other groups (control, IR alone and 10mg/kg for 6 days).

Comparison of survival curves:

log rank (Mantel-Co) test: chi-square 34.39; df3; p value <0.0001; p-value summary there was a significant difference in survival curves.

Log rank test of trend: chi-square 6.590; df1; p value 0.0103; p value summary; a significant trend.

Gehan-Breslow-Wilcoxon test: chi-square 28.79; df3; p value <0.0001; p value summary; there was a significant difference in survival curves.

Results from the GL261 mouse model are shown in figure 2. As shown in fig. 2, the results from the continuous administration of 10mg/kg SP are significantly different from the control and IR alone.

Comparison of survival curves:

log rank (Mantel-Co) test: chi-square 21.32; df3; p value <0.0001; p value summary; there was a significant difference in survival curves.

Log rank test of trend: chi-square 17.42; df 1; p value <0.0001; p value summary; a significant trend.

Gehan-Breslow-Wilcoxon test: chi-square 16.37; df3; p value <0.0010; p value summary; there was a significant difference in survival curves.

Example 2

Orthotopic GL261 tumors were established on day 0. Starting on day 7, mice were treated with 60mg/kg Temozolomide (TMZ) for 5 days +/-10 mg/kg SP orally administered starting on day 7 until the end of the study. The results are shown in fig. 3. Figure 3 shows that mice receiving tmz+sp have improved survival relative to mice receiving TMZ alone. Future animal studies will be combined with fractionated radiation therapy.

Example 3

The new object recognition test is a behavioral test in which a mouse is caused to explore two similar objects during a first (training) period, and then after a certain length of time, one of the two training objects is replaced with a new object during a later (testing) period. Because mice have an innate preference for novelty, if they recognize familiar objects, they will spend most of their time on new objects. The amount of time spent exploring new objects provides a measure of cognitive memory.

24 adult female C57BL/6J mice were treated as follows: (1) 8 mice were Irradiated (IR) with a total dose of 10Gy divided into three parts with a "rest" day between parts covering about 1 square millimeter of the center of the head; (2) 8 mice were similarly irradiated and also treated with 10 mg/kg/day of aminopterin (SP), which was administered as an oral gavage, starting 6 days after the 1 st to last irradiated portion of IR (i.e., 11 days total); (3) naive 8 mice untreated.

Mice were tested in a New Object Recognition (NOR) task at 1 hour interval between trials (ITI) to assess hippocampal mediated memory 1 month after treatment (i.e., testing occurred one hour after training). During the training and testing phase, the time to investigate an object was measured to be 5 minutes. Two independent observers were used for evaluation. Discrimination index= (novel-familiar)/(novel + familiar). As shown in fig. 4, administration of SP increased the discrimination index compared to the irradiation group alone.

Single factor analysis of variance:

normalization test (shape-Wilk): through (p=0.992)

Equal variance test (Brown-Forsythe): pass (p=0.540).

Group name	N	Omission of	Average value of	Standard deviation of	SEM
						Young person	8	0	0.371	0.120	0.0423
IR	8	0	0.127	0.193	0.0682
						IR+ drugs	8	0	0.262	0.227	0.0801

Variant source	DF	SS	MS	F	P
						Inter-group	2	0.240	0.120	3.500	0.049
Residual error	21	0.720	0.0343
						Sum total	23	0.960

All pairwise multiple comparison procedure (Tukey test):

factor comparison: treatment group

Comparison of	Average difference	p	q	P	P<0.050
						Relative to IR of naive species	0.244	3	3.735	0.039	Is that
Naive versus ir+ drugs	0.110	3	1.674	0.475	Whether or not
						IR+ drug vs IR	0.135	3	2.061	0.331	Whether or not

Mice were tested for spontaneous alternation in the y maze 2 months after treatment. At 3 months post-treatment, mice were retested for NOR at 1 hour ITI.

Example 4: RNA study

Progressive, late-onset damage to the brain following high doses of radiation is thought to be caused by radiation-induced persistent free radicals, reactive oxygen species, and pro-inflammatory cytokines. ICAM-1, TNF- α, IL-1 β and IFN- γ are all up-regulated in the mouse brain after whole brain irradiation and are involved in the development of late lesions in the brain. RNA extracts from brain tissue were obtained on days 8, 30 and 60 after 10Gy (3 fxs x 3.3 Gy) whole brain irradiation (TBI) with and without 10mg/kg SP for 6 days and analyzed for the above cytokines. As we have previously demonstrated in lung tissue, we expect that expression of these pro-inflammatory cytokines increases after TBI exposure and that treatment with SP will normalize their expression.

Example 5: phase 2 study of Murray in combination with temozolomide in recent diagnosis or recurrent glioblastoma

Summary of the inventionsummary

This is an open-label, randomized phase 2 clinical trial that tests the safety and efficacy of aminopterin (SP) for the treatment of newly diagnosed or recurrent glioblastoma. The study design is shown in fig. 5. The aim of this study was to test the safety profile of aminopterin plus standard Temozolomide (TMZ) chemotherapy in patients with newly diagnosed or recurrent glioblastoma and establish Recommended Doses (RD) for phase 3. Study will test and standard TMZ chemoradiotherapy groupsThe combined 3 methotrexate dose levels (20, 40, or 60 mg/kg/day) were compared to standard TMZ chemotherapy alone. Patients randomized to aminopterin were treated at 20 mg/kg/day, 40 mg/kg/day, or 60 mg/kg/day for 6 days ("induction treatment" in fig. 5) as part of induction treatment. This is followed by standard therapy (the "radiation therapy" in fig. 5) consisting of: 60Gy/30 radiation therapy was continued for 6 weeks with 75mg/m daily ² TMZ, with respect to the noted group of aminopterin, was used to add aminopterin. Followed by a 4 week disruption of TMZ treatment, with only the duration of the treatment of aminopterin ("TMZ disruption" in FIG. 5), followed by maintenance treatment with 6 TMZ 150-200mg/m on days 1 to 5 of q28 ² Maintenance cycles combined with daily administration of the aminopterin per 28-day chemotherapy maintenance cycle ("maintenance therapy" in fig. 5). Patients randomized to TMZ alone will be treated with standard therapy (60 Gy/30 radiation therapy plus 75mg/m per day for 6 weeks from 60 Gy/30) for each 28-day chemotherapy maintenance period ² TMZ followed by a 4 week interruption of TMZ treatment followed by a 150-200mg/m TMZ with 6 days 1 to 5 of q28 ² Maintenance therapy composition of maintenance cycle) of the patient. See study protocol of fig. 5.

Target object

Main outcome metrics

Secondary outcome measure of 6 month Progression Free Survival (PFS) rate for all participants by RECIST version 1.1

Overall Response Rate (ORR) for all patients by RECIST version 1.1; OOR was assessed by conducting study imaging every 6-9 weeks after the first dose of study treatment. ORR is defined as the proportion of participants in the analysis population that have a Complete Response (CR), defined as the disappearance of all target lesions, a decrease in the minor axis of the pathological lymph nodes to <10mm, or a Partial Response (PR), defined as the decrease of at least 30% of the sum of target lesion diameters using the baseline total diameter as a reference.

Disease control rate for all patients by RECIST version 1.1; disease control rate is defined as the proportion of patients whose best curative effect achieves complete remission, partial remission, or disease control is maintained for at least 4 weeks by RECIST version 1.1.

Overall Survival (OS) of all participants; OS is defined as the time from the first day of study treatment to death due to any cause, and the truncated data was analyzed by the Kaplan-Meier method and reported monthly.

Median PFS of all participants; median PFS is defined as the median time from the first day of study treatment to the first recording of disease Progression (PD) according to RECIST version 1.1 or death (based on the first occurrence) for any reason.

Median OS for all participants, defined as the time from the first day of study treatment to death due to any cause.

Security as measured by Adverse Events (AEs) including Serious Adverse Events (SAE).

Criteria for

Inclusion criteria:

1. voluntary participation and written informed consent

2. Subject is greater than or equal to 18 years old

3. The space-occupying lesion on the curtain is diagnosed as glioblastoma through pathology;

4. patients were scheduled for standard radiation therapy and temozolomide concurrent chemotherapy after surgery

Mri confirmed a clear recurrence of the newly diagnosed or tumor. For recurrence, the diameter of the enhancement focus is over 1cm and more than 2 layers (layer spacing 5 mm), or recurrence is confirmed by pathology after a biopsy or surgery again;

6. According to RECIST version 1.1, there is at least one measurable lesion;

karnofsky functional status scale (KPS) score > 60 points;

8. the method can swallow the aminopterin or TMZ normally;

9. the expected survival time exceeds 3 months;

10. sufficient organ and bone marrow function. The definition is as follows.

a. Neutrophil Count (ANC) greater than or equal to 1500/mm ³ (1.5×10 ⁹ /L)；

b. Platelet count (PLT) is not less than 100000/mm ³ (100×10 ⁹ /L)；

c. Hemoglobin (HB) is more than or equal to 9g/dl (90 g/L);

d. serum albumin is more than or equal to 2.8g/dl;

e. serum creatinine is not more than 1.5 times of the upper limit value (ULN) of normal value or creatinine clearance is not less than 60ml/min;

f. total Bilirubin (TB) is less than or equal to 1.5 XULN, or Total Bilirubin (TB) is greater than 1.5 XULN, but Direct Bilirubin (DBIL) is less than or equal to 1 XULN; patients with liver metastases should be +.2XULN;

g. the level of aspartate Aminotransferase (AST)/serum glutamate-oxaloacetate transaminase (SGOT) or alanine transaminase (ALT)/Serum Glutamate Pyruvate Transaminase (SGPT) should be less than or equal to 2.5×uln and in patients with liver metastasis should be less than or equal to 5×uln;

h. left Ventricular Ejection Fraction (LVEF) > 50%, corrected Q-T interval (QTc) in men <450ms and in women <470ms;

11. in patients not receiving anticoagulant therapy, the international normalized ratio of prothrombin time (INR) is 1.5 or less and the Activated Partial Thromboplastin Time (APTT) is 1.5 times the upper limit of the normal value or less. Patients receiving full dose or parenteral anticoagulant therapy may enter clinical trials as long as the dose of anticoagulant drug is stable for at least 2 weeks prior to entering the clinical study and the results of the clotting test are within the limits of local treatment;

12. Women of childbearing age should have a negative pregnancy test (serum or urine) during the first 7 days of participation and voluntarily use appropriate contraceptive methods during the observation period and 8 weeks after the last administration of study medication; for men, the appropriate contraceptive method should be surgically sterilized or agreed to be used during the observation period and within 8 weeks after the last study drug administration;

13. good compliance, and can be matched with research and follow-up according to program requirements.

Exclusion criteria:

1. prior history of allergy to temozolomide or aminopterin;

2. major surgery (permission of biopsy for diagnosis; permission of tumor resection if possible) or severe trauma within 4 weeks prior to the first study medication;

3. other clinical studies are currently being engaged in unless it is an observational (non-intervention) clinical study or a follow-up intervention for a new clinical study; or to take into account any other drug clinical study within 4 weeks prior to the first administration, or no more than 5 half-lives from the last study medication;

4. basal cell carcinoma or squamous cell carcinoma, superficial bladder carcinoma, cervical carcinoma in situ, intraductal carcinoma in situ, and papillary thyroid carcinoma of the skin that can be treated topically and that has been cured in the past 5 years or simultaneously;

5. Advanced patients (including patients with uncontrollable large amounts of exudates [ chest, pericardial cavity, abdominal cavity ]) who are symptomatic, diffuse to the viscera and are at risk of life threatening complications in a short period of time;

6. fever of unknown origin of >38.5 ℃ occurred during the screening period/prior to the first administration (tumor-induced fever may be included in the group at the discretion of the investigator);

7. serious infections (CTCAE grade > 2) such as severe pneumonia, bacteremia, infectious complications, etc. occur within 4 weeks prior to first use study medication, active lung inflammation revealed by baseline chest imaging examination, symptoms and signs of infection within 2 weeks prior to first use study medication, or require oral or intravenous antibiotic treatment (prophylactic use of antibiotics is excluded)

8. The following occurred within 6 months prior to study entry: myocardial infarction, severe/unstable angina, new York Heart Association (NYHA) grade 2 or above cardiac insufficiency and clinically significant supraventricular or ventricular arrhythmias, require clinical intervention; hypertension with poor drug control (systolic blood pressure continuously rises to be more than or equal to 150mmhg or diastolic blood pressure is more than or equal to 100 mmhg);

9. history of gastrointestinal bleeding or tendency to gastrointestinal bleeding in the past 6 months, such as esophageal varices, localized active ulcer lesions, fecal occult blood ++ (+) (gastroscopy is required when fecal occult blood is (+);

10. Inability to swallow study drugs, chronic diarrhea (including but not limited to irritable bowel syndrome, crohn's disease, ulcerative colitis), ileus, and other factors affecting drug administration and absorption;

11. allogeneic organ transplantation or allogeneic hematopoietic stem cell transplantation history or congenital immunodeficiency are known;

12. patients with active tuberculosis infection are found through medical history or CT examination, or patients with active tuberculosis infection in the previous year, or patients with active tuberculosis infection history but not treated periodically before one year;

13. human Immunodeficiency Virus (HIV) infection or acquired immunodeficiency syndrome (AIDS); untreated active hepatitis B (hepatitis B is defined as positive test result of hepatitis B virus surface antigen [ HBsAg ], HBV-DNA is not less than 500, hepatitis C is defined as positive hepatitis C antibody [ HCV AB ], HCV-RNA is higher than detection limit of analytical method and abnormal liver function) or combined hepatitis B and hepatitis C combined infection;

14. patients have a well-defined history of neurological or psychiatric disorders, including history of epilepsy and dementia, and are known to have a history of substance abuse, alcoholism or substance abuse;

15. is considered unsuitable for the patients studied.

16. Concomitant use of methotrexate or other antifolates.

Other embodiments are within the claims.

Claims

1. A method of treating glioblastoma in a subject, the method comprising administering to the subject an effective amount of methotrexate or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein the effective amount of the aminopterin or pharmaceutically acceptable salt thereof is from about 10mg/kg to about 60mg/kg per dose.

3. The method of claim 1, wherein the effective amount of the aminopterin or pharmaceutically acceptable salt thereof is from about 20mg/kg to about 60mg/kg per dose.

4. The method of claim 1, wherein the effective amount of the aminopterin or pharmaceutically acceptable salt thereof is about 20mg/kg per dose.

5. The method of claim 1, wherein the effective amount of the aminopterin or pharmaceutically acceptable salt thereof is about 40mg/kg per dose.

6. The method of claim 1, wherein the effective amount of the aminopterin or pharmaceutically acceptable salt thereof is about 60mg/kg per dose.

7. The method of any one of claims 1 to 6, wherein the effective amount of the aminopterin or pharmaceutically acceptable salt thereof is administered once daily.

8. The method of any one of claims 1 to 6, wherein the effective amount of the aminopterin or pharmaceutically acceptable salt thereof is administered twice daily.

9. The method of claim 8, wherein the effective amount of the aminopterin or pharmaceutically acceptable salt thereof is administered in two equal doses.

10. The method of any one of claims 1 to 9, wherein the effective amount of the aminopterin or pharmaceutically acceptable salt thereof is administered with a food item.

11. The method of claim 10, wherein administration to the subject occurs less than 30 minutes prior to or after consumption of the food.

12. The method of claim 10, wherein the administration to the subject is substantially concurrent with food.

13. The method of any one of claims 10 to 12, wherein the food is a high protein and/or high fat food.

14. The method of any one of claims 10 to 12, wherein the food is a low fat food.

15. The method of any one of claims 10 to 12, wherein the food is a high calorie food.

16. The method of any one of claims 1 to 9, wherein the effective amount of the aminopterin or pharmaceutically acceptable salt thereof is administered in the absence of food.

17. The method of claim 16, wherein administration to the subject occurs more than 30 minutes prior to eating the food or more than 2 hours after eating the food.

18. The method of claim 16, wherein administration to the subject occurs more than 30 minutes prior to eating the food or more than 3 hours after eating the food.

19. The method of any one of claims 1 to 18, wherein the methotrexate or pharmaceutically acceptable salt thereof is formulated as an oral powder for suspension.

20. The method of any one of claims 1 to 18, wherein the methotrexate or pharmaceutically acceptable salt thereof is administered as a suspension in a flavored suspension vehicle.

21. The method of any one of claims 1 to 18, wherein the methotrexate or pharmaceutically acceptable salt thereof is administered as a powder suspended in water or juice.

22. The method of any one of claims 1 to 21, further comprising treating the subject with therapeutic radiation.

23. The method of claim 22, wherein the aminopterin or pharmaceutically acceptable salt thereof is administered for the duration of the therapeutic radiation.

24. The method of any one of claims 22 to 23, wherein the methotrexate or pharmaceutically acceptable salt thereof is administered prior to the therapeutic radiation.

25. The method of any one of claims 22 to 24, wherein the methotrexate or pharmaceutically acceptable salt thereof is administered after the therapeutic radiation.

26. The method of any one of claims 1 to 25, wherein the methotrexate or pharmaceutically acceptable salt thereof is administered for at least 6 days.

27. The method of any one of claims 1 to 25, wherein the methotrexate or pharmaceutically acceptable salt thereof is administered for at least 14 days.

28. The method of any one of claims 1 to 25, wherein the methotrexate or pharmaceutically acceptable salt thereof is administered for at least 30 days.

29. The method of any one of claims 1 to 28, wherein the methotrexate or pharmaceutically acceptable salt thereof is administered at least 14 days after the therapeutic radiation.

30. The method of any one of claims 1 to 28, wherein the methotrexate or pharmaceutically acceptable salt thereof is administered at least 30 days after the therapeutic radiation.

31. The method of any one of claims 1 to 30, wherein the methotrexate or pharmaceutically acceptable salt thereof is administered sequentially simultaneously with the therapeutic radiation.

32. The method of any one of claims 1 to 31, wherein the methotrexate or pharmaceutically acceptable salt thereof is administered continuously over a 28 day period.

33. The method of any one of claims 1 to 31, wherein the methotrexate or pharmaceutically acceptable salt thereof is administered for a period of six 28 days.

34. The method of claim 33, wherein the aminopterin or pharmaceutically acceptable salt thereof is administered for a period of more than six 28 days.

35. The method of any one of claims 1 to 34, wherein the subject is a human.

36. The method of any one of claims 1 to 35, wherein the method further comprises treating the subject with Temozolomide (TMZ).